Methods of making hypermutable cells using PMSR homologs

ABSTRACT

Methods of making cells hypermutable are disclosed using PMS2 homologs that have a common sequence motif. The PMS2 homologs of the invention have ATPase-like motifs and are at least about 90% identical to PMS2-134. Methods of generating mutant libraries and using the PMS2 homologs in diagnostic and therapeutic applications for cancer are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/358,578, filed Feb. 21, 2002, the disclosure of whichis incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

[0002] The invention is related to the area of mismatch repair genes. Inparticular it is related to the field of generating hypermutable cellsusing dominant negative mismatch repair genes wherein the proteinsencoded by the mismatch repair gene comprise a consensus sequence for anATPase.

BACKGROUND OF THE INVENTION

[0003] Within the past four years, the genetic cause of the HereditaryNonpolyposis Colorectal Cancer Syndrome (HNPCC), also known as Lynchsyndrome II, has been ascertained for the majority of kindred's affectedwith the disease (Liu, B. et al. (1996) Nat. Med. 2:169-174). Themolecular basis of HNPCC involves genetic instability resulting fromdefective mismatch repair (MMR). To date, six genes have been identifiedin humans that encode for proteins and appear to participate in the MMRprocess, including the mutS homologs GTBP, hMSH2, hMSH3 and the mutLhomologs hMLH1, hPMSI, and hPMS2 (Bronner, C. E. et al. (1994) Nature368:258-261; Fishel, R. et al. (1993) Cell 7:1027-1038; Leach, F. S. etal. (1993) Cell 75:1215-1225; Nicolaides, N. C., et al. (1994) Nature371:75-80; Nicolaides, N. C. et al. (1996) Genomics 31:395-397; Palombo,F. et al. (1995) Science 268:1912-1914; Papadopoulos, N. et al. (1994)Science 263:1625-1629). Mutations or epigenetic changes affecting thefunction of these genes have been reported for all of the homologslisted above in tumor tissues exhibiting microsatellite instability(MI), a type of genomic instability that results from “slippagemutations” in mono-, di, or tri-nucleotide repeats due to MMR deficiency(Jiricny, J., and M. Nystrom-Lahti (2000) Curr. Opin. Genet. Dev.10:157-161; Perucho, M. (1996) Biol. Chem. 377:675-684; Strand, M. etal. (1993) Nature 365:274-276). While germline mutations in all of thesegenes have been identified in HNPCC kindreds (Bronner, C. E. et al.(1994) Nature 368:258-261; Leach, F. S. et al. (1993) Cell 75:1215-1225;Liu, B. et al. (1996) Nat. Med. 2:169-174; Nicolaides, N. C. et al.(1994) Nature 371:75-80; Papadopoulos, N. et al. (1994) Science263:1625-1629), many examples exist where tumor types exhibiting MI lackmutations in any of the known MMR genes, suggesting the presence ofadditional genes that are involved in the MMR process (personalobservation; Nagy, M. et al. (2000) Leukemia 14:2142-2148; Peltomaki P.(2001) Hum. Mol. Genet. 10:735-740; Wang Y. et al. (2001) Int. J. Cancer93:353-360). In addition to its occurrence in virtually all tumorsarising in HNPCC patients, MI is also found in a subset of sporadictumors with distinctive molecular and phenotypic properties originatingfrom many different tissue types, suggesting a role for an expandedinvolvement of defective MMR in other cancer types (Nagy, M. et al.(2000) Leukemia 14:2142-2148; Peltomaki P. (2001) Hum. Mol. Genet.10:735-740; Wang Y. et al. (2001) Int. J. Cancer 93:353-360; StarostikP. et al. (2000) Am. J. Pathol. 157:1129-1136; Chen Y. et al. (2001)Cancer Res. 61:4112-4121).

[0004] Though the mutator defect that arises from the MMR deficiency canaffect any DNA sequence, microsatellite sequences are particularlysensitive to MMR abnormalities (Modrich, P. (1994) Science266:1959-1960). Microsatellite instability (MI) is therefore a usefulindicator of defective MMR. In addition to its occurrence in virtuallyall tumors arising in HNPCC patients, MI is found in a small fraction ofsporadic tumors with distinctive molecular and phenotypic properties(Perucho, M. (1996) Biol. Chem. 377:675-684).

[0005] HNPCC is inherited in an autosomal dominant fashion, so that thenormal cells of affected family members contain one mutant allele of therelevant MMR gene (inherited from an affected parent) and one wild-typeallele (inherited from the unaffected parent). During the early stagesof tumor development, however, the wild-type allele is inactivatedthrough a somatic mutation, leaving the cell with no functional MMR geneand resulting in a profound defect in MMR activity. Because a somaticmutation in addition to a germ-line mutation is required to generatedefective MMR in the tumor cells, this mechanism is generally referredto as one involving two hits, analogous to the biallelic inactivation oftumor suppressor genes that initiate other hereditary cancers (Leach, F.S. et al. (1993) Cell 75:1215-1225; Liu, B. et al. (1996) Nat. Med.2:169-174; Parsons, R. et al. (1993) Cell 75:1227-1236). In line withthis two-hit mechanism, the non-neoplastic cells of HNPCC patientsgenerally retain near normal levels of MMR activity due to the presenceof the wild-type allele.

[0006] Genetic studies have unequivocally shown that inactivation ofmismatch repair (MMR) genes, including PMS2, results in geneticinstability and tumorigenesis in human and rodent tissues. In themajority of cases, inactivation of both alleles of a particular MMR geneare required to completely knockout a component of the MMR spell checksystem, a process that is similar to the “two-hit” hypothesis forinactivation of tumor suppressor alleles. Independent studies focused onscreening for mutated MMR genes in normal and neoplastic tissues haveconfirmed the two hit hypothesis except for 2 cases where only a singlemutated allele of a MMR gene was found associated in tumors. This alleleis a PMS2 gene containing a nonsense mutation at codon 134, whichresults in a truncated polypeptide that encodes for a 133 amino acidprotein capable of eliciting a dominant negative effect on the MMRactivity of the cell. This hypothesis was confirmed by subsequentstudies demonstrating the ability of the PMS134 protein to cause adominant negative effect on the MMR activity of an otherwise MMRproficient mammalian cell.

[0007] The truncated domain of PMS134 is highly homologous to the codingregion of PMSR2 and PMSR3 proteins, sharing an identity of greater than90% at the protein level. However, PMSR2 and PMSR3 do not appear to beexpressed in normal tissues and have not been shown to be associatedwith HNPCC.

[0008] The ability to alter the signal transduction pathways bymanipulation of a gene products function, either by over-expression ofthe wild type protein or a fragment thereof, or by introduction ofmutations into specific protein domains of the protein, the so-calleddominant-negative inhibitory mutant, were described over a decade in theyeast system Saccharomyces cerevisiae by Herskowitz (1987) Nature 329(6136):219-222). It has been demonstrated that over-expression of wildtype gene products can result in a similar, dominant-negative inhibitoryphenotype due most likely to the “saturating-out” of a factor, such as aprotein, that is present at low levels and necessary for activity;removal of the protein by binding to a high level of its cognate partnerresults in the same net effect, leading to inactivation of the proteinand the associated signal transduction pathway. Recently, work done byNicolaides et al. (Nicolaides N. C. et al. (1998) Mol. Cell. Biol.18:1635-1641; U.S. Pat. No. 6,146,894 to Nicolaides et al.) hasdemonstrated the utility of introducing dominant negative inhibitorymismatch repair mutants into mammalian cells to confer global DNAhypermutability. The ability to manipulate the MMR process, andtherefore, increase the mutability of the target host genome at will, inthis example a mammalian cell, allows for the generation of innovativecell subtypes or variants of the original wild type cells. Thesevariants can be placed under a specified, desired selective process, theresult of which is a novel organism that expresses an altered biologicalmolecule(s) and has a new trait. The concept of creating and introducingdominant negative alleles of a gene, including the MMR alleles, inbacterial cells has been documented to result in genetically alteredprokaryotic mismatch repair genes (Aronshtam A. and M. G. Marinus (1996)Nucl. Acids Res. 24:2498-2504; Wu T. H. and M. G. Marinus (1994) J.Bacteriol. 176:5393-400; Brosh R. M. Jr. and S. W. Matson (1995) J.Bacteriol. 177:5612-5621).

[0009] Furthermore, altered MMR activity has been demonstrated when MMRgenes from different species including yeast, mammalian cells, andplants are over-expressed (Fishel, R. et al. (1993) Cell 7:1027-1038;Studamire B. et al. (1998) Mol. Cell. Biol. 18:75907601; Alani E. et al.(1997) Mol. Cell. Biol. 17:2436-2447; Lipkin S. M. et al. (2000) Nat.Genet. 24:27-35).

[0010] Recently Guarne et al. (2001) EMBO J. 20(19):5521-5531 describedthe ATPase function of the MutLα, a heterodimer of MLH1 and PMS2. Guarneet al. studied the three dimensional structure of PMS2 and determinedthe portions of the molecule that participate in ATP binding andhydrolysis. Guarne et al. postulate that dimerization and ATPaseactivity are probably required for MMR function. Guarne et al., however,do not teach or suggest how their findings relate to dominant negativephenotypes of mismatch repair.

[0011] There is a continuing need in the art for methods of geneticallymanipulating cells to increase their performance characteristics andabilities. To this end, there is a need in the art to understand,develop and design MMR genes that confer a dominant negative effect foruse in generating hypermutable cells.

SUMMARY OF THE INVENTION

[0012] The invention provides methods of making a cell hypermutablecomprising introducing into the cell a PMS2 homolog comprising anucleotide sequence encoding a polypeptide comprising the amino acidsequence of SEQ ID NO:23, thereby making the cell hypermutable, whereinthe PMS2 homolog is other than PMSR2 and PMSR3.

[0013] The invention also provides methods of making a mutation in agene of interest comprising introducing into a cell containing a gene ofinterest a PMS2 homolog comprising a nucleotide sequence encoding apolypeptide wherein the polypeptide comprises the amino acid sequence ofSEQ ID NO:23, thereby making said cell hypermutable, wherein the PMS2homolog is other than PMSR2 and PMSR3, and selecting a mutant cellcomprising a mutation in said gene of interest.

[0014] The invention also provides methods of making dominant negativeMMR genes for introduction into cells to create hypermutable cells. Thedominant negative MMR genes encode proteins comprising the amino acidsequence of SEQ ID NO:23 and share at least about 90% homology withPMS2-134 (SEQ ID NO:13).

[0015] The invention also provides methods of generating libraries ofmutated genes. In embodiments of the methods of the invention, adominant negative allele of a PMS2 homolog is introduced into a cellwhereby the cell becomes hypermutable. The cells accumulate mutations ingenes and a population of cells may therefore comprise a library ofmutated genes as compared to wild-type cells with a stable genome.

[0016] In some embodiments of the methods of the invention, thepolypeptides comprise the amino acid sequence of SEQ ID NO:24. In someembodiments, the polypeptides have a conserved ATPase domain. In someembodiments of the method of the invention the PMS2 homolog is a PMSR6.In certain embodiments of the method of the invention, the PMSR6polypeptide comprises the amino acid sequence of SEQ ID NO:22 and isencoded by the polynucleotide sequence of SEQ ID NO:21.

[0017] In some embodiments of the methods of the invention, the PMS2homolog further comprises a truncation which results in an inability todimerize with MLH1. This may be a truncation from the E′ α-helix to theC-terminus, the E α-helix to the C-terminus, the F α-helix to theC-terminus, the G α-helix to the C-terminus, the H′ α-helix to theC-terminus, the H α-helix to the C-terminus, or the I α-helix to theC-terminus, for example, as described by Guarne et al. (2001) EMBO J.20(19):5521-5531 and shown in FIG. 2.

[0018] The methods of the invention may be used for eukaryotic cells,particularly cells from protozoa, yeast, insects, vertebrates, andmammals, particularly humans. The methods of the invention may also beused for prokaryotic cells, such as bacterial cells, and may be used forplant cells.

[0019] The methods of the invention may also include treating the cellswith a chemical mutagen or radiation to increase the rate of mutationover that observed by disrupting mismatch repair alone.

[0020] The hypermutable cells of the invention may be screened to detecta mutation in a gene of interest that confers a desirable phenotype. Thecells may be screened by examining the nucleic acid, protein or thephenotype of the cells.

[0021] In some embodiments of the methods of the invention, geneticstability may be restored to the hypermutable cells, thereby maintainingcells comprising mutations in the gene of interest which may be furtherfaithfully propagated.

[0022] The invention also provides methods of assaying cells to detectneoplasia comprising contacting said sample with a nucleotide sequenceencoding the amino acid sequence of SEQ ID NO:23 to detect expression ofa polynucleotide encoding a PMS2 homolog comprising the amino acidsequence of SEQ ID NO:23, wherein expression of said PMS2 homolog isassociated with neoplasia. The detecting of the PMS2 homolog may beaccomplished by any means known in the art, including but not limited toNorthern blot analysis and RT-PCR.

[0023] The invention also provides methods of assaying cells to detectneoplasia comprising contacting said sample with an antibody directedagainst a PMS2 homolog or peptide fragments thereof; and detecting thepresence of an antibody-complex formed with the PMS2 homolog or peptidefragment thereof, thereby detecting the presence of said PMS2 homolog insaid sample, wherein the presence of said PMS2 homolog is associatedwith neoplasia. Methods of detection of PMS2 homologs may be by anymeans known in the art, including but not limited to radioimmunoassays,western blots, immunofluorescence assays, enzyme-linked immunosorbentassays (ELISA), and chemiluminescence assays.

[0024] The invention also provides methods of treating a patient withcancer comprising identifying a patient with a PMS2 homolog-associatedneoplasm, administering to said patient an inhibitor of expression ofsaid PMS2 homolog wherein said inhibitor suppresses expression of saidPMS2 homolog in said PMS2 homolog associated neoplasm. Such neoplasmsinclude, for example, lymphomas. Inhibitors of PMS2 homolog expressioninclude antisense nucleotides, ribozymes, antibody fragments and ATPaseanalogs that specifically bind the PMS2 homolog.

BRIEF DESCRIPTION OF THE FIGURES

[0025]FIG. 1 shows the polypeptide sequences of PMSR2, PMSR3 and PMSR6showing consensus sequence regions with underlining. FIG. 1B shows analignment of the consensus sequence region of PMS2 with a DNAgyrase-like ATPase motif.

[0026]FIG. 2A and B show the structure of the N-terminal fragment ofPMS2 (orthagonal views) and FIG. 2C shows a sequence alignment of hPMS2,hMLH1 and MutL N-terminal fragments and structural featurescorresponding to FIGS. 2A and B (from Guarne et al. (2001) EMBO J.20(19):5521-5531, FIG. 2A-C).

[0027]FIG. 3 shows RT-PCR analysis of PMSR genes in lymphoma cell lines.Thirty cycles of RT-PCR amplification was performed on lymphoma celllines with (lanes 3-5) or without (lane 2) microsatellite instability(MI). As demonstrated above, each line with MI expressed either thePMSR2 or the PMSR3 gene, while no expression was observed in cell lineslacking MI (lane 2). hPMS2 and β-actin message was used as internalcontrols to measure for RNA loading. Lane 1 was a mock reaction tomeasure for potential artifact or contamination. Additional PCRamplifications were performed using 45 cycles of amplification whichresulted in more robust products in positive lanes, as observed with 30cycles, while no PMSR signal was detected in negative samples such asthose presented in lanes 1 and 2.

[0028]FIG. 4 shows Western blot analysis of human lymphoma cell lineswith (LMM-1) (lane 2) or without (LNM-a) (lane 1) microsatelliteinstability (MI). The arrows indicate proteins with the expectedmolecular weight of the hPMS2 and hPMSR2 polypeptides. A correlation ofPMSR expression is observed in lymphoma cell lines exhibiting MI.

[0029]FIG. 5 shows β-galactosidase activity in 293 cells expressing PMS2and PMSR homologs plus the MMR-sensitive pCAR-OF reporter. Cells inwhich MMR activity is decreased results in MI leading toinsertion-deletion mutations within the β-galactosidase gene, a subsetof which will restore the open reading frame (ORF) and producefunctional enzyme. Cells are grow for 17 days and then harvested forprotein lysates to measure β-galactosidase activity generated by eachcell line. Cells in which a high rate of mutagenesis has occurred willproduce β-galactosidase activity, while cells in which MMR activity isfunctional will retain background levels of enzymatic activity. Eachcell line was tested in two independent experiments (experiment 1 andexperiment 2). Extracts were incubated with a calorimetric galactosesubstrate for 1 hour. Enzyme activity as a function of substrateconversion was measured by optical density at 576 nm as described(Nicolaides, N. C. et al. (1998) Mol. Cell. Biol. 18:1635-1641). Asshown above, cells expressing PMSR2 and PMSR3 had a high degree of MIleading to an increase in β-galactosidase activity. MI was monitored atthe gene level to confirm that genetic alterations occurred within thepolynucleotide repeat disrupting the β-galactosidase ORF.

DETAILED DESCRIPTION OF THE INVENTION

[0030] The present invention provides methods of making cellshypermutable using derivatives of mismatch repair genes bearing aconsensus sequence for an ATPase. The consensus sequence is present in anumber of PMS2 homologs that confers a dominant negative phenotype ofmismatch repair when transfected into host cells.

[0031] PMS2 homologs, such as PMSR2 and PMSR3 encode homologs of themutL mismatch repair family of proteins. Both PMSR2 and PMSR3 proteins,for example, are highly homologous to the N-terminus of the human PMS2gene and its encoded polypeptide. Functional studies have shown thatwhen the PMSR2 or PMSR3 cDNAs are expressed in MMR proficient mammaliancells either of these homologs are capable of inactivating MMR in adominant negative fashion resulting in genetic instability (see FIG. 5).

[0032] Preliminary gene expression studies have found that the PMSR2 orPMSR3 genes are not expressed in non-neoplastic tissues and are onlydetected in a subset of human lymphoma cell lines, of Burkitt's lymphomaorigin, that exhibit microsatellite instability, a hallmark of MMRdeficiency (see FIGS. 3 and 4).

[0033] The present invention is directed to use of PMS2 homologs whichcomprise the conserved domain of PMS134, PMSR2 and PMSR3 and shareconserved portions of ATPase domains for use in generating hypermutablecells by introducing into cells polynucleotide sequences encoding PMS2homologs which function to decrease MMR activity in a dominant negativefashion.

[0034] It has been discovered that proteins comprising a consensussequence and homology to the N-terminal domain of PMS2, includingstructural features of ATPase domains function as dominant negativemismatch repair inhibitors. In a specific embodiment, PMSR6 expressionconfers a dominant negative phenotype of MMR deficiency in cells.

[0035] It has further been discovered that proteins comprising theconsensus sequence of SEQ ID NO:23 or SEQ ID NO:24 and comprising aportion having at least about 90% homology with PMS2-134 can confer adominant negative phenotype and a reduction in MMR activity whenintroduced into cells. In some embodiments the PMS2 homologs compriseATPase domains. The PMS2 homologs may further comprise domains that areother than MMR proteins, such as chimeric or fusion proteins comprisinga domain that is homologous to PMS2-134 and a portion that isheterologous.

[0036] As used herein, the term “PMS2 homolog” refers to a polypeptidesequence having the consensus sequence of AVKE LVENSLDAGA TN (SEQ IDNO:23). In some embodiments, the PMS2 homologs comprise the polypeptidesequence of LRPNAVKE LVENSLDAGA TNVDLKLKDY GVDLIEVSGN GCGVEEENFE (SEQ IDNO:24). The PMS2 homologs comprise this structural feature and, whilenot wishing to be bound by any particular theory of operation, it isbelieved that this structural feature correlates with ATPase activitydue to the high homology with known ATPases. The knowledge of thisstructural feature and correlated function and the representative numberof examples provided herein, will allow one of ordinary skill in the artto readily identify which proteins may be used in the methods of theinvention.

[0037] As used herein a “nucleic acid sequence encoding a PMS2 homolog”refers to a nucleotide sequence encoding a polypeptide having the ATPaseconsensus sequence motifs and that, when expressed in a cell decreasesthe activity of mismatch repair in the cell. The nucleic acid sequencesencoding the PMS2 homologs, when introduced and expressed in the cells,increase the rate of spontaneous mutations by reducing the effectivenessof endogenous mismatch repair mediated DNA repair activity, therebyrendering the cell highly susceptible to genetic alterations, (i.e.,render the cells hypermutable). Hypermutable cells can then be utilizedto screen for mutations in a gene or a set of genes in variant siblingsthat exhibit an output trait(s) not found in the wild-type cells. ThePMS2 homologs may be an altered mismatch repair genes, or may be amismatch repair gene that when overexpressed in the cell results in animpaired mismatch repair activity.

[0038] The nucleic acid sequences encoding the PMS2 homologs areintroduced into the cells and expressed. The cell's mismatch repairactivity is decreased and the cell becomes hypermutable. In someembodiments, the cells may be further incubated with a chemical mutagento further enhance the rate of mutation.

[0039] While it has been documented that MMR deficiency can lead to asmuch as a 1000-fold increase in the endogenous DNA mutation rate of ahost, there is no assurance that MMR deficiency alone will be sufficientto alter every gene within the DNA of the host bacterium to createaltered biochemicals with new activity(s). Therefore, the use ofchemical mutagens and their respective analogues such as ethidiumbromide, EMS, MNNG, MNU, Tamoxifen, 8-Hydroxyguanine, as well as otherssuch as those taught in: Khromov-Borisov, N. N., et al. (1999) Mutat.Res. 430:55-74); Ohe, T. et al. (1999) Mutat. Res. 429:189-199); Hour,T. C. et al. (1999) Food Chem. Toxicol. 37:569-579); Hrelia, P. et al.(1999) Chem. Biol. Interact. 118:99111); Garganta, F. et al. (1999)Environ. Mol. Mutagen. 33:75-85); Ukawa-Ishikawa S. et al. (1998) Mutat.Res. 412:99-107; www.ehs.utah.edu/ohh/mutagens, etc. can be used tofurther enhance the spectrum of mutations and increase the likelihood ofobtaining alterations in one or more genes that can in turn generatehost cells with a desired new output trait(s). Mismatch repairdeficiency leads to hosts with an increased resistance to toxicity bychemicals with DNA damaging activity. This feature allows for thecreation of additional genetically diverse hosts when mismatch defectivecells are exposed to such agents, which would be otherwise impossibledue to the toxic effects of such chemical mutagens [Colella, G. et al.(1999) Br. J. Cancer 80:338-343); Moreland, N. J. et al. (1999) CancerRes. 59:2102-2106); Humbert, O. et al. (1999) Carcinogenesis20:205-214); Glaab, W. E. et al. (1998) Mutat. Res. 398:197-207].

[0040] The cells that may be transfected with the PMS2 homologs includeany prokaryotic or eukaryotic cell. The prokaryotic cells may bebacterial cells of a wide array of genera.

[0041] In other embodiments, the cells are eukaryotic cells, such as,but not limited to insect cells, protozoans, yeast, fungi, vertebratecells (such as, for example, fish, avian, reptilian and amphibiancells), mammalian cells (including, for example, human, non-humanprimate, rodent, caprine, equine, bovine, and ovine cells).

[0042] In other embodiments, plant cells may be transfected with a PMS2homolog to render the plant cells hypermutable.

[0043] Once cells are rendered hypermutable, the genome of the cellswill begin to accumulate mutations, including mutations in genes ofinterest. The mutations in the genes of interest may confer upon thesegenes desirable new phenotypes that can be selected. As a non-limitingexample, mutations in protein-encoding genes may render the proteinsexpressed at higher levels. As another non-limiting example, proteinssuch as antibodies and enzymes may have altered binding characteristics,such as higher affinities for their antigen or substrate, respectively.Such altered phenotypes may be screened and the cells containing thegenes and displaying the altered phenotypes may be selected for furthercultivation.

[0044] The genome of the cells containing the genes of interest with newphenotype may be rendered genetically stable by counteracting theeffects of the transfected PMS2 homologs. Those of skill in the art may“cure” the cells of plasmids that contain the PMS2 homologs or disruptthe PMS2 homolog within the cell such that the PMS2 homolog is no longerexpressed. Plasmids that are maintained in cells only under drugpressure may be used to cultivate the cells with PMS2 homologs. When thedrug pressure is removed the cells tend to lose the plasmids. In otherembodiments, inducible expression vectors may be used to express thePMS2 homologs. Thereafter, the inducer molecule may be withdrawn toallow the genome to stabilize.

[0045] As used herein, the term “mismatch repair,” also called “mismatchproofreading,” refers to an evolutionarily highly conserved process thatis carried out by protein complexes described in cells as disparate asprokaryotic cells such as bacteria to more complex mammalian cells(Modrich, P. (1994) Science 266:1959-1960; Parsons, R. et al. (1995)Science 268:738-740; Perucho, M. (1996) Biol Chem. 377: 675-684). Amismatch repair gene is a gene that encodes one of the proteins of sucha mismatch repair complex. Although not wanting to be bound by anyparticular theory of mechanism of action, a mismatch repair complex isbelieved to detect distortions of the DNA helix resulting fromnon-complementary pairing of nucleotide bases. The non-complementarybase on the newer DNA strand is excised, and the excised base isreplaced with the appropriate base that is complementary to the olderDNA strand. In this way, cells eliminate many mutations that occur as aresult of mistakes in DNA replication, resulting in genetic stability ofthe sibling cells derived from the parental cell.

[0046] Some wild type alleles as well as dominant negative alleles causea mismatch repair defective phenotype even in the presence of awild-type allele in the same cell. An example of a dominant negativeallele of a mismatch repair gene is the human gene hPMS2-134, whichcarries a truncation mutation at codon 134 (Parsons, R. et al. (1995)Science 268:738-740; Nicolaides N. C. et al (1998) Mol. Cell. Biol.18:1635-1641). The mutation causes the product of this gene toabnormally terminate at the position of the 134^(th) amino acid,resulting in a shortened polypeptide containing the N-terminal 133 aminoacids. Such a mutation causes an increase in the rate of mutations,which accumulate in cells after DNA replication. Expression of adominant negative allele of a mismatch repair gene results in impairmentof mismatch repair activity, even in the presence of the wild-typeallele. Any PMS2 homolog, which produces such effect, can be used inthis invention, whether it is wild-type or altered, whether it derivesfrom mammalian, yeast, fungal, amphibian, insect, plant, bacteria or isdesigned as a chimera or fusion protein.

[0047] Yeast, for example, which may be the source of host MMR, may bemutated or not. The term “yeast” used in this application comprises anystrain from the eukaryotic kingdom, including but not limited toSaccharomyces sp., Pichia sp., Schizosaccharomyces sp., Kluyveromycessp., and other fungi (Gellissen, G. and Hollenberg, C. P. (1997) Gene190(1):87-97). These organisms can be exposed to chemical mutagens orradiation, for example, and can be screened for defective mismatchrepair. Genomic DNA, cDNA, mRNA, or protein from any cell encoding amismatch repair protein can be analyzed for variations from thewild-type sequence. Dominant negative alleles of PMS2 homologs can alsobe created artificially, for example, by creating fusion proteins orchimeric proteins in which a portion of the protein comprises theconsensus sequence of SEQ ID NO:23 or SEQ ID NO:24, has about 90% aminoacid homology with PMS2-134, and another portion that is a heterologousamino acid sequence.

[0048] Various techniques of site-directed mutagenesis can be used. Thesuitability of such alleles, whether natural or artificial, for use ingenerating hypermutable yeast can be evaluated by testing the mismatchrepair activity (using methods described in Nicolaides N. C. et al.(1998) Mol. Cell. Biol. 18:1635-1641) caused by the allele in thepresence of one or more wild-type alleles to determine if it is adominant negative allele.

[0049] A cell that over-expresses a wild type mismatch repair allele ora dominant negative allele of a mismatch repair gene will becomehypermutable. This means that the spontaneous mutation rate of such cellis elevated compared to cells without such alleles. The degree ofelevation of the spontaneous mutation rate can be at least 2-fold,5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, or1000-fold that of the normal cell as measured as a function of celldoubling/hour.

[0050] According to one aspect of the invention, a polynucleotideencoding the PMS2 homolog is introduced into a cell such as a mammaliancell, vertebrate cell, plant cell, or yeast, for example. The gene is aPMS2 homolog and is a dominant negative. The PMS2 homolog can benaturally occurring or made in the laboratory. The polynucleotide can bein the form of genomic DNA, cDNA, RNA, or a chemically synthesizedpolynucleotide or polypeptide. The molecule can be introduced into thecell by transformation, electroporation, mating, particle bombardment,or other method described in the literature.

[0051] Transformation is used herein as any process whereby apolynucleotide or polypeptide is introduced into a cell. The process oftransformation can be carried out in a yeast culture using a suspensionof cells.

[0052] In general, transformation will be carried out using a suspensionof cells but other methods can also be employed as long as a sufficientfraction of the treated cells incorporate the polynucleotide orpolypeptide so as to allow transfected cells to be grown and utilized.The protein product of the polynucleotide may be transiently or stablyexpressed in the cell. Techniques for transformation are well known tothose skilled in the art. Available techniques to introduce apolynucleotide or polypeptide into a cell include but are not limited toelectroporation, viral transduction, cell fusion, the use ofspheroplasts or chemically competent cells (e.g., calcium chloride), andpackaging of the polynucleotide together with lipid for fusion with thecells of interest. Once a cell has been transformed with the mismatchrepair gene or protein, the cell can be propagated and manipulated ineither liquid culture or on a solid agar matrix, such as a petri dish.If the transfected cell is stable, the gene will be expressed at aconsistent level for many cell generations, and a stable, hypermutableyeast strain results.

[0053] An isolated yeast cell can be obtained from a yeast culture bychemically selecting strains using antibiotic selection of an expressionvector. If the yeast cell is derived from a single cell, it is definedas a clone. Techniques for single-cell cloning of microorganisms such asyeast are well known in the art.

[0054] A polynucleotide encoding a PMS2 homolog can be introduced intothe genome of yeast or propagated on an extra-chromosomal plasmid, suchas the 2-micron plasmid. Selection of clones harboring a mismatch repairgene expression vector can be accomplished by plating cells on syntheticcomplete medium lacking the appropriate amino acid or other essentialnutrient as described (Schneider, J. C. and L. Guarente (1991) Methodsin Enzymology 194:373). The yeast can be any species for which suitabletechniques are available to produce transgenic microorganisms, such asbut not limited to genera including Saccharomyces, Schizosaccharomyces,Pichia, Hansenula, Kluyveromyces and others.

[0055] Any method for making transgenic yeast known in the art can beused. According to one process of producing a transgenic microorganism,the polynucleotide is introduced into the yeast by one of the methodswell known to those in the art. Next, the yeast culture is grown underconditions that select for cells in which the polynucleotide encodingthe mismatch repair gene is either incorporated into the host genome asa stable entity or propagated on a self-replicating extra-chromosomalplasmid, and the protein encoded by the polynucleotide fragmenttranscribed and subsequently translated into a functional protein withinthe cell. Once transgenic yeast is engineered to harbor the expressionconstruct, it is then propagated to generate and sustain a culture oftransgenic yeast indefinitely.

[0056] Once a stable, transgenic cell has been engineered to express aPMS2 homolog, the cell can be cultivated to create novel mutations inone or more target gene(s) of interest harbored within the same cell. Agene of interest can be any gene naturally possessed by the cell or oneintroduced into the cell host by standard recombinant DNA techniques.The target gene(s) may be known prior to the selection or unknown. Oneadvantage of employing transgenic yeast cells to induce mutations inresident or extra-chromosomal genes within the yeast is that it isunnecessary to expose the cells to mutagenic insult, whether it ischemical or radiation, to produce a series of random gene alterations inthe target gene(s). This is due to the highly efficient nature and thespectrum of naturally occurring mutations that result as a consequenceof the altered mismatch repair process. However, it is possible toincrease the spectrum and frequency of mutations by the concomitant useof either chemical and/or radiation together with MMR defective cells.The net effect of the combination treatment is an increase in mutationrate in the genetically altered yeast that are useful for producing newoutput traits. The rate of the combination treatment is higher than therate using only the MMR-defective cells or only the mutagen withwild-type MMR cells. The same strategy is useful for other types ofcells including vertebrate and mammalian cells.

[0057] MMR-defective cells of the invention can be used in geneticscreens for the direct selection of variant sub-clones that exhibit newoutput traits with commercially desirable applications. This permits oneto bypass the tedious and time-consuming steps of gene identification,isolation and characterization.

[0058] Mutations can be detected by analyzing the internally and/orexternally mutagenized cells for alterations in its genotype and/orphenotype. Genes that produce altered phenotypes in MMR-defectivemicrobial cells can be discerned by any of a variety of moleculartechniques well known to those in the art. For example, the cell genomecan be isolated and a library of restriction fragments of the yeastgenome can be cloned into a plasmid vector. The library can beintroduced into a “normal” cell and the cells exhibiting the novelphenotype screened. A plasmid can be isolated from those normal cellsthat exhibit the novel phenotype and the gene(s) characterized by DNAsequence analysis.

[0059] Alternatively, differential messenger RNA screen can be employedutilizing driver and tester RNA (derived from wild type and novelmutant, respectively) followed by cloning the differential transcriptsand characterizing them by standard molecular biology methods well knownto those skilled in the art. Furthermore, if the mutant sought isencoded by an extra-chromosomal plasmid, then following co expression ofthe dominant negative MMR gene and the gene of interest, and followingphenotypic election, the plasmid can be isolated from mutant clones andanalyzed by DNA sequence analysis using methods well known to those inthe art.

[0060] Phenotypic screening for output traits in MMR-defective mutantscan be by biochemical activity and/or a readily observable phenotype ofthe altered gene product. A mutant phenotype can also be detected byidentifying alterations in electrophoretic mobility, DNA binding in thecase of transcription factors, spectroscopic properties such as IR, CD,X-ray crystallography or high field NMR analysis, or other physical orstructural characteristics of a protein encoded by a mutant gene. It isalso possible to screen for altered novel function of a protein in situ,in isolated form, or in model systems. One can screen for alteration ofany property of the yeast associated with the function of the gene ofinterest, whether the gene is known prior to the selection or unknown.

[0061] The screening and selection methods discussed are meant toillustrate the potential means of obtaining novel mutants withcommercially valuable output traits, but they are not meant to limit themany possible ways in which screening and selection can be carried outby those of skill in the art.

[0062] Plasmid expression vectors that harbor a PMS2 homolog insert canbe used in combination with a number of commercially availableregulatory sequences to control both the temporal and quantitativebiochemical expression level of the dominant negative MMR protein. Theregulatory sequences can be comprised of a promoter, enhancer orpromoter/enhancer combination and can be inserted either upstream ordownstream of the MMR gene to control the expression level. Theregulatory sequences can be any of those well known to those in the artfor extra-chromosomal expression vectors or on constructs that areintegrated into the genome via homologous recombination. These types ofregulatory systems have been disclosed in scientific publications andare familiar to those skilled in the art.

[0063] Once a cell with a novel, desired output trait of interest iscreated, the activity of the aberrant MMR activity is desirablyattenuated or eliminated by any means known in the art. These includebut are not limited to removing an inducer from the culture medium thatis responsible for promoter activation, curing a plasmid from atransformed yeast cell, and addition of chemicals, such as 5-fluoroorotic acid to “loop-out” the gene of interest.

[0064] In the case of an inducibly controlled dominant negative PMS2homolog, expression of the PMS2 homolog will be turned on (induced) togenerate a population of hypermutable cells with new output traits.Expression of the dominant negative MMR allele can be rapidly turned offto reconstitute a genetically stable strain that displays a new outputtrait of commercial interest. The resulting cell is now useful as astable cell line that can be applied to various commercial applications,depending upon the selection process placed upon it.

[0065] In cases where genetically deficient mismatch repair cell areused to derive new output traits, transgenic constructs can be used thatexpress wild type mismatch repair genes sufficient to complement thegenetic defect and therefore restore mismatch repair activity of thehost after trait selection [Grzesiuk, E. et al. (1998) Mutagenesis13:127-132); Bridges, B. A. et al. (1997) EMBO J. 16:3349-3356);LeClerc, J. E. (1996) Science 15:1208-1211); Jaworski, A. et al. (1995)Proc. Natl. Acad. Sci USA 92:11019-11023]. The resulting cell isgenetically stable and can be employed for various commercialapplications.

[0066] The use of over-expression of foreign (exogenous, transgenic)mismatch repair genes from human and yeast such as MSH2, MLH1, MLH3,etc. have been previously demonstrated to produce a dominant negativemutator phenotype in yeast hosts (Shcherbakova, P. V. et al. (2001) Mol.Cell. Biol. 21(3):940-951; Studamire, B. et al. (1998) Mol. Cell. Biol.18:7590-7601; Alani E. et al. (1997) Mol. Cell. Biol. 17:2436-2447;Lipkin, S. M. et al. (2000) Nat. Genet. 24:27-35). In addition, the useof yeast strains expressing prokaryotic dominant negative MMR genes aswell as hosts that have genomic defects in endogenous MMR proteins havealso been previously shown to result in a dominant negative mutatorphenotype (Evans, E. et al. (2000) Mol. Cell. 5(5):7897-7899; AronshtamA. and M. G. Marinus (1996) Nucl. Acids Res. 24:2498-2504; Wu, T. H. andM. G. Marinus (1994) J. Bacteriol. 176:5393-5400; Brosh R. M. Jr., andS. W. Matson (1995) J. Bacteriol. 177:5612-5621). However, the findingsdisclosed here teach the use of PMS2 homologs, including the human PMSR2gene (Nicolaides, N. C. et al. (1995) Genomics 30:195-206), the relatedPMS2-134 truncated MMR gene (Nicolaides N. C. et al. (1995) Genomics29:329-334), the plant mismatch repair genes (U.S. patent applicationSer. No. 09/749,601) and those genes that are homologous to the 134N-terminal amino acids of the PMS2 gene to create hypermutable yeast.

[0067] The ability to create hypermutable organisms using PMS2 homologscan be used to generate innovative yeast strains that display new outputfeatures useful for a variety of applications, including but not limitedto the manufacturing industry, for the generation of new biochemicals,for detoxifying noxious chemicals, either by-products of manufacturingprocesses or those used as catalysts, as well as helping in remediationof toxins present in the environment, including but not limited topolychlorobenzenes (PCBs), heavy metals and other environmental hazards.Novel cell lines can be selected for enhanced activity to either produceincreased quantity or quality of a protein or non-protein therapeuticmolecule by means of biotransformation. Biotransformation is theenzymatic conversion of one chemical intermediate to the nextintermediate or product in a pathway or scheme by a microbe or anextract derived from the microbe. There are many examples ofbiotransformation in use for the commercial manufacturing of importantbiological and chemical products, including penicillin G, erythromycin,and clavulanic acid. Organisms that are efficient at conversion of “raw”materials to advanced intermediates and/or final products also canperform biotransformation (Berry, A. (1996) Trends Biotechnol.14(7):250-256). The ability to control DNA hypermutability in host cellsusing a PMS2 homolog allows for the generation of variant subtypes thatcan be selected for new phenotypes of commercial interest, including butnot limited to organisms that are toxin-resistant, have the capacity todegrade a toxin in situ or the ability to convert a molecule from anintermediate to either an advanced intermediate or a final product.

[0068] Other applications using PMS2 homologs to produce geneticalteration of host cells for new output traits include but are notlimited to recombinant production strains that produce higher quantitiesof a recombinant polypeptide as well as the use of altered endogenousgenes that can transform chemical or catalyze manufacturing downstreamprocesses. A regulatable PMS2 homolog can be used to produce a cell witha commercially beneficial output trait. Using this process, cellsexpressing a PMS2 homolog can be directly selected for the phenotype ofinterest. Once a selected cell with a specified output trait isisolated, the hypermutable activity can be turned-off by several methodswell known to those skilled in the art. For example, if the PMS2 homologis expressed by an inducible promoter system, the inducer can be removedor depleted. Such systems include but are not limited to promoters suchas: lactose inducibleGALi-GAL10 promoter (Johnston, M. and R. W. Davis(1984) Mol. Cell Biol. 4:1440); the phosphate inducible PH05 promoter(Miyanohara, A. et al. (1983) Proc. Natl. Acad. Sci. USA 80:1-5); thealcohol dehydrogenase I (ADH) and 3-phosphoglycerate kinase (PGK)promoters, that are considered to be constitutive but can berepressed/de-repressed when yeast cells are grown in non-fermentablecarbon sources such as but not limited to lactate (Ammerer, G. (1991)Methods in Enzymology 194:192; Mellor, J. et al. (1982) Gene 24:563);Hahn S. and L. Guarente (1988) Science 240:317); Alcohol oxidase (AOX)in Pichia pastoris (Tschopp, J. F. et al (1987) Nucl. Acids Res.15(9):3859-76; and the thiamine repressible expression promoter nmtl inSchizosaccharomyces pombe (Moreno, M. B. et al. (2000) Yeast16(9):861-872). Yeast cells can be transformed by any means known tothose skilled in the art, including chemical transformation with LiCI(Mount, R. C. et al. (1996) Methods Mol. Biol. 53:139-145) andelectroporation (Thompson, J. R. et al. (1998) Yeast 14(6):565-571).Yeast cells that have been transformed with DNA can be selected forgrowth by a variety of methods, including but not restricted toselectable markers (URA3; Rose, M. et al. (1984) Gene 29:113; LEU2;Andreadis, A. et al. (1984) J. Biol. Chem. 259:8059; ARG4; Tschumper G.and J. Carbon (1980) Gene 10:157; and HIS3; Struhl, K. et al. (1979)Proc. Natl. Acad. Sci. USA 76:1035) and drugs that inhibit growth ofyeast cells (tunicamycin, TUN; Hahn, S. et al. (1988) Mol. Cell Biol.8:655). Recombinant DNA can be introduced into yeast as described aboveand the yeast vectors can be harbored within the yeast cell eitherextra-chromosomally or integrated into a specific locus.Extra-chromosomal based yeast expression vectors can be either high copybased (such as the 2-pm vector Yep13; Rose, A. B. and J. R. Broach(1991) Methods in Enzymology 185:234), low copy centromeric vectors thatcontain autonomously replicating sequences (ARS) such as YRp7(Fitzgerald-Hayes, M. et al. (1982) Cell 29:235) and well as integrationvectors that permit the gene of interest to be introduced into specifiedlocus within the host genome and propagated in a stable manner(Rothstein, R. J. (1991) Methods in Enzymology 101:202). Ectopicexpression of MMR genes in yeast can be attenuated or completelyeliminated at will by a variety of methods, including but not limited toremoval from the medium of the specific chemical inducer (e.g., depletegalactose that drives expression of the GAL10 promoter in Saccharomycescerevisiae or methanol that drives expression of the AOX1 promoter inPichia pastoris), extrachromosomally replicating plasmids can be “cured”of expression plasmid by growth of cells under non-selective conditions(e.g., YEp13 harboring cells can be propagated in the presence ofleucine,) and cells that have genes inserted into the genome can begrown with chemicals that force the inserted locus to “loop-out” (e.g.,integrants that have URA3 can be selected for loss of the inserted geneby growth of integrants on 5-fluoroorotic acid (Boeke, J. D. et al.(1984) Mol. Gen. Genet. 197:345-346). Whether by withdrawal of induceror treatment of yeast cells with chemicals, removal of MMR expressionresults in the reestablishment of a genetically stable yeast cell-line.Thereafter, the lack of mutant MMR allows the endogenous, wild type MMRactivity in the host cell to function normally to repair DNA. The newlygenerated mutant yeast strains that exhibit novel, selected outputtraits are suitable for a wide range of commercial processes or forgene/protein discovery to identify new biomolecules that are involved ingenerating a particular output trait. Of course, yeast is only oneexample of cell types that may be used and similar strategies usingknown promoters and inducers may be employed for use in other types ofcells including vertebrate, insect, and mammalian cells, for example.

[0069] Moreover, mismatch repair is responsible for repairingchemically-induced DNA adducts, therefore blocking this process couldtheoretically increase the number, types, mutation rate and genomicalterations of a yeast [Rasmussen, L. J. et al. (1996) Carcinogenesis17:2085-2088); Sledziewska Gojska, E. et al. (1997) Mutat. Res.383:31-37); and Janion, C. et al. (1989) Mutat. Res. 210:15-22)]. Inaddition to the chemicals listed above, other types of DNA mutagensinclude ionizing radiation and UV irradiation, which is known to causeDNA mutagenesis in yeast, can also be used to potentially enhance thisprocess (Lee C. C. et al (1994) Mutagenesis 9:401-405; Vidal A. et al.(1995) Carcinogenesis 16:817-821). These agents, which are extremelytoxic to host cells and therefore result in a decrease in the actualpool size of altered yeast cells are more tolerated in MMR defectivehosts and in turn permit an enriched spectrum and degree of genomicmutagenesis.

[0070] The general methods of the invention therefore also provide amethod of generating libraries of mutated genes in which the cells madehypermutable from the introduction of the PMS2 homologs accumulatemutations and may be used subsequently to produce cDNA and genomiclibraries comprising mutated genes (as compared to the wild-typeparental host cells). Methods of preparing cDNA and genomic librariesare well known in the art and techniques may be found, for example inSambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, Third Edition,2001.

[0071] The invention also provides methods of assaying cells to detectneoplasia comprising contacting said sample with a nucleotide sequenceencoding the amino acid sequence of SEQ ID NO:23 to detect expression ofa polynucleotide encoding a PMS2 homolog comprising the amino acidsequence of SEQ ID NO:23, wherein expression of said PMS2 homolog isassociated with neoplasia.

[0072] The PMS2 homolog is identified as having the consensus sequenceof SEQ ID NO:23 or SEQ ID NO:24 and may be detected by nucleic acidscomprising a sequence that encodes SEQ ID NO:23 or SEQ ID NO:24. One ofordinary skill in the art may design reverse transcriptase-polymerasechain reaction assays (RT-PCR assays) to detect the expression of thePMS2 homologs in the cells suspected of being neoplastic. Northern blotsmay also be used to detect PMS2 homolog expression using standardprotocols such as those found in, for example, Sambrook et al. MOLECULARCLONING: A LABORATORY MANUAL, Third Edition, 2001.

[0073] The invention also provides methods of assaying cells to detectneoplasia comprising contacting said sample with an antibody directedagainst a PMS2 homolog or peptide fragments thereof; and detecting thepresence of an antibody-complex formed with the PMS2 homolog or peptidefragment thereof, thereby detecting the presence of said PMS2 homolog insaid sample, wherein the presence of said PMS2 homolog is associatedwith neoplasia. Methods of detection of PMS2 homologs may be by anymeans known in the art, including but not limited to radioimmunoassays,western blots, immunofluorescence assays, enzyme-linked, immunosorbentassays (ELISA), and chemiluminescence assays. The various protocols forthese assays are well-known in the art.

[0074] The invention also provides methods of treating a patient withcancer comprising identifying a patient with a PMS2 homolog-associatedneoplasm, administering to said patient an inhibitor of expression ofsaid PMS2 homolog wherein said inhibitor suppresses expression of saidPMS2 homolog in said PMS2 homolog associated neoplasm. Such neoplasmsinclude, for example, lymphomas. Inhibitors of PMS2 homolog expressioninclude antisense nucleotides, ribozymes, antibody fragments and ATPaseanalogs that specifically bind the PMS2 homolog.

[0075] The antisense molecules are polynucleotides that arecomplementary to a portion of the RNA encoding the PMS2 homolog and bindspecifically to the RNA. The antisense molecules inhibit the translationof the PMS2 homolog RNA and thereby inhibit the effect of PMS2expression. Antisense molecules may be directed to portions of the RNAthat are involved in robosome binding or initiation of translation aswell as to portions of the coding sequence. Generally antisensemolecules are at least 15 nucleotides in length, but may be 20, 25, 30,35, 40, 45, 50 or more nucleotides in length.

[0076] Ribozymes are a special catalytic class of antisense moleculesthat cleave substrate nucleotides. Design of ribozymes for PMS2 homologsmay be performed using methods well-known in the art, as described, forexample in Lyngstadaas S P. (2001) “Synthetic hammerhead ribozymes astools in gene expression” Crit. Rev. Oral. Biol. Med. 12(6):469-78;Samarsky D, Ferbeyre G, Bertrand E. (2000) “Expressing active ribozymesin cells” Curr. Issues Mol. Biol. 2(3):87-93. The ribozyme or vectorencoding a ribozyme are introduced into the cells expressing the PMS2homolog and are activated such that the ribozyme binds to and cleavesthe polynucleotide encoding the PMS2 homolog, thereby preventingexpression of the PMS2 homolog.

[0077] The above disclosure generally describes the present invention. Amore complete understanding can be obtained by reference to thefollowing specific examples that will be provided herein for purposes ofillustration only, and are not intended to limit the scope of theinvention.

EXAMPLES Example 1 Evaluation The Association Of PMSR2 And PMSR3 RNAExpression In Tumors Of Lymphoid Tissue and Comparison WithMicrosatellite Instability Profile

[0078] A panel of lymphoma tissues and cell lines are analyzed formicrosatellite instability (MI) by PCR mediated genotypic analysis andfor PMSR2 and PMSR3 expression via RT-PCR analysis following methodspreviously used and described in publications by Dr. Nicolaides (Liu, B.et al. (1996) Nature Med. 2:169-174; Nicolaides, N. C. et al. (1996)Genomics 31:395-397). For RNA expression studies, RNAs are extractedfrom a panel of 83 lymphoma cell lines (obtained from ATCC and personalcontacts) using the trizol method as described by the manufacturer(Gibco/BRL). 100 ngs of total RNA are reverse transcribed usingSuperscriptII reverse transcriptase (RT) and random hexamers as primerin 20 μl reactions as recommended by the manufacturer (Gibco/BRL). Eachsample is incubated in reaction buffer with (RT+) or without (RT−) RT,where the RT− samples serve as negative control. Reactions are incubatedfor 1 hour at 37° C. and diluted to a final volume of one hundredmicroliters. Routinely, 5 μls of each sample is used for PCRamplification in 25 μl reactions containing 67 mM Tris, pH 8.8, 16.6 mM(NH₄)₂SO₄, 6.7 mM MgCl₂, 10 mM 2-mercaptoethanol, 4% DMSO, 1.25 mM eachof the four dNTPs, 175 ng of each cDNA specific primer and 1 U of Taqpolymerase. Amplifications are carried out at 94° C. for 30 sec, 58° C.for 90 sec, 72° C. for 90 sec for 30 cycles. One half of the reaction isloaded onto 1% agarose gels in 1×Tris Acetate EDTA running buffer anddetected by ethidium bromide staining. Below is a table (Table 1) withthe gene specific primers and expected molecular weight PCR fragments.Samples are scored positive if an RT+ reaction contains a DNA fragmentof the expected molecular weight while no signal is observed in RT− orwater controls. TABLE 1 Primers for specific amplification of PMSR cDNAsfrom cells and tissues. Gene Forward primer Reverse primer Size (bp)hPMS2 5′-ggacgagaagtataacttcgag-3′ 5′-catctcgcttgtgttaagagc-3′ 372 (SEQID NO: 27) (SEQ ID NO: 28) hPMSR2 5′-ggcgcaaccaaagcaagag-3′5′-actgcgttttttccgaacg-3′ 221 (SEQ ID NO: 29) (SEQ ID NO: 30) hPMSR35′-atgttggagaactacagcc-3′ 5′-cactccatagtccttaagc-3′ 278 (SEQ ID NO: 31)(SEQ ID NO: 32) β-actin 5′-gggaatgggtcagaaggac-3′5′-tttcacggttggccttaggg-3′ 209 (SEQ ID NO: 33) (SEQ ID NO: 34)

[0079] Cell lines already determined to express PMSR2 and PMSR3 are usedas positive controls while lines previously identified as PMSR null areused as negative controls. Samples are analyzed in duplicates to confirmreproducibility of expression.

[0080] To assess for microsatellite instability of lymphoma samples,DNAs are isolated from a panel of lymphomas as described above. DNAswill be isolated using the proteinase K digestion and phenol extractionprocedure as described (Liu et al. (1996) Nature Med.2:169-174). Variousamounts of test DNAs from lymphoma cells and HCT116 (a MMR defectivehuman colon epithelial cell line) are used to determine the sensitivityof our microsatellite test. The D2S123, BAT26, and BAT40 alleles areknown to be heterogeneous in HCT116 cells and are therefore used as apositive control for detection of MI. To measure for MI, DNAs aretitrated by limiting dilution to determine the level of sensitivity foreach marker set. DNAs are PCR amplified using the BAT26F:5′-tgactacttttgacttcagcc-3′ (SEQ ID NO:35) and the BAT26R:5′-aaccattcaacatttttaaccc-3′ (SEQ ID NO:36); BAT40F:5′-attaacttcctacaccacaac-3′ (SEQ ID NO:37) and BAT40R:5′-gtagagcaagaccaccttg-3′ (SEQ ID NO:38); and D2S123F:5′-acattgctggaagttctggc-3′ (SEQ ID NO:39) and D2S123R:5′-cctttctgacttggatacca-3′ (SEQ ID NO:40) primers in buffers asdescribed (Nicolaides, N. C., et al. (1995) Genomics 30:195-206).Briefly, 1 pg to 100 ngs of DNA is amplified using the followingconditions: 94° C. for 30 sec, 50-55° C. for 30 sec, 72° C. for 30 secfor 30 cycles. PCR reactions are then resolved on 8% denaturingpolyacrylamide gels and visualized by autoradiography. Preliminarystudies using these reagents and DNA extracted from paraffin-embeddedtissues routinely find that 0.1 ng of genomic DNA is the limit ofdetection using our conditions.

[0081] Microsatellite stability may be measured in cells using twentyindependent reactions of 0.01 ngs of DNA from the same clinical sampleor cells by PCR. This concentration typically allows for the measurementof 1 genome equivalent per sample and allows for the detection ofmicrosatellite alterations in clonal variants that have occurred duringthe growth of a particular cell line or tissue. Samples are scored MI+if at least two samples of a particular marker are found to have PCRfragments that differ from the predominate allele size for a givensample. Statistical analysis is performed by comparing the number of MI+cells expressing PMSR2 or PMSR3 with those not expressing either PMSRgene.

Example 2 Generation Of Polyclonal Antisera Specific For PMSR2 And PMSR3For Immunostaining And Proof Of Concept At The Protein Level

[0082] The ability to produce antibodies that can specifically recognizePMSR2 or PMSR3 is of great utility for establishing methods for in situanalysis of tissues expressing these proteins as diagnostic markers. Asdemonstrated in FIG. 4, the generation of PMSR-specific peptides is usedfor tissue analysis to determine specific expression of a particularPMSR polypeptide. The immunoblot shown in FIG. 4 demonstrates the needfor new antisera that allows for the specific detection of a PMSRprotein without cross-reactivity to other PMS homologs. To generate PMSRspecific antisera, we will synthesize 20 amino acid peptides and couplethem to KLH immunogen for antisera production in rabbits. Peptides thatare directed to the amino and carboxy termini of the hPMSR2 and hPMSR3proteins may be generated by known methods. The amino acid sequences ofthe peptides to be synthesized are provided in Table 2. All peptides aredirected to the first or last 20 amino acid residues of the encodedpolypeptide (Nicolaides, N. C. et al. (1998) Mol. Cell. Biol.18:1635-1641), except for the N-terminal hPMSR3 peptide which containsamino acids 5 to 26 to avoid multiple cysteine and tryptophan residueswhich have posed solubility problems for our group in the past. TABLE 2Peptides for PMSR2 and PMSR3 specific antisera Protein N-terminalpeptide C-terminal peptide hPMSR2 MAQPKQERVARARHQRSETALEDNVITVFSSVKNGPGSSR (SEQ ID NO:41) (SEQ ID NO:42) hPMSR3RPRLGRRCMVSPRARAPREQ GVEEENFEGLISFSSETSHM (SEQ ID NO:43) (SEQ ID NO:44)

[0083] The peptides produced are purified and analyzed by MassSpectroscopy and HPLC analysis. 3 mgs of immunopure peptide areconjugated to keyhole limpet haemocyanin (KLH) carrier using awater-soluble carbodiimide, which eliminates the need for a cysteineresidue in the sequence. The remaining peptide material is used forantisera analysis by ELISA and western blot. After conjugation, theKLH-linked peptide is resuspended in Freund's adjuvant and is ready forimmunization.

[0084] Rabbits are immunized against each peptide using the followingprotocol. At Day 0, a prebleed will be taken from each host rabbit.Antigen is administered to rabbits by an injection of a solutioncontaining adjuvant on a weekly schedule with three scheduled bleeds atday 49, 63, and 77, where a 20 ml sample of serum is collected andanalyzed. Bleeds will be analyzed for antisera directed againstimmunizing peptides for PMSR2 and PMSR3 by Enzyme Linked Immuno-SorbantAssay (ELISA) and western blots.

[0085] ELISA assays are performed to test antibody titer in unpurifiedbleeds to measure for antibody reactivity to native peptides describedabove. Briefly, 96 well plates are coated with 50 uls of a 1 ug/mlsolution containing each peptide for 4 hours at 4° C. Wells containingeach peptide are probed by each antiserum to measure for background andantibody specificity. Plates are washed 3 times in calcium and magnesiumfree phosphate buffered saline solution (PBS^(−/−)) and blocked in 100uls of PBS^(−/−) with 5% dry milk for 1 hour at room temperature. Afterblocking, wells are rinsed and incubated with 100 uls of a PBS solutioncontaining a 1:5 dilution of preimmune serum or respective bleeds fromeach rabbit for 2 hours. Plates are then washed 3 times with PBS^(−/−)and incubated for 1 hour at room temperature with 50 uls of a PBS^(−/−)solution containing 1:3000 dilution of a sheep anti-rabbit horseradishperoxidase (HRP) conjugated secondary antibody. Plates are then washed 3times with PBS^(−/−) and incubated with 50 uls of TMB-HRP substrate(BioRad) for 15 minutes at room temperature to detect antibody titers.Reactions are stopped by adding 50 uls of 500 mM sodium bicarbonate andanalyzed by OD at 415 nm using a BioRad plate reader. Samples aredetermined to be positive if an enhanced signal over background(preimmune serum and/or negative control peptides) are observed.

[0086] Western blot are also performed using antisera generated above asa probe to demonstrate the ability of antisera to recognize the expectedmolecular weight protein in whole cell extracts. First, unconjugatedpeptides are tested for antibody reactivity. The peptides listed inTable 2 are added to 20 μs of 2×SDS lysis buffer (60 mM Tris, pH 6.8/2%SDS/0.1 M 2-mercaptoethanol/0.1% bromophenol blue) and boiled for 2 min.Twenty microliters of each sample is then electrophoresed in 18%Tris-glycine SDS/PAGE gels for 10 minutes and electroblotted ontoImmobilon-P (Millipore) membrane in transfer buffer (48 mM Tris/40 mMglycine/0.0375% SDS/20% methanol) for 20 minutes to maximize peptidebinding. Filters are blocked overnight in blocking buffer (TBS, 0.05%Tween-20/5% powdered milk). Filters are probed with different prebleedsand antiserum from each rabbit followed by a secondary horseradishperoxidase conjugated anti-rabbit (Pierce) and prepared forchemiluminescence. Samples are deemed positive if the appropriateantisera reacts with the corresponding peptide antigen while no reactionis observed in negative or peptide control lanes. Samples are alsodeemed positive if no reaction is observed using preimmune serum.

[0087] The activity of positive antisera as described above is analyzedusing whole cell lysates in western blot using extracts from cellspreviously identified to express PMSR2 and PMSR3 at the RNA and/or inthe case of PMSR2, at the protein level (which is recognized byanti-PMS2 antisera, see FIG. 4). Fifty thousand cells are centrifugedand directly lysed in 25 μl of 2×sample buffer and boiled for 5 minutes.Samples are loaded on 4-20% Tris-glycine gels and electroblotted asdescribed above except electrophoresis and transfer time is 1 hour.Filters are probed with various antisera and bleed lots and detected asabove. Antisera are deemed positive if immunoreactions are observed inPMSR positive lines but are absent in PMSR negative cell lines. Positivereactions will be further confirmed for specificity by monitoring forendogenous PMS2 cross-reactivity as seen in FIG. 4 as well ascompetition using various peptides to monitor for binding. If backgroundis observed in any antiserum, reaction conditions are altered bychanging blocking buffers, washing stringencies, and dilution ofantisera, parameters that have been routinely modified by our group forsuccessful antibody probing.

[0088] PMSR specific antiserum may be purified using Pierce Igpurification kits, for example, that are able to purify totalimmunoglobulin to >95% purity. Antibody totals are quantitated byspectrophotometry, resuspended at a concentration of 1 mg/ml in PBScontaining sodium azide as preservative. Antisera are re-tested foractivity in western blot using 1:10, 1:100, and 1:1000 dilution todetermine optimal concentration of pure materials. Purified antisera maythen be used for immunohistological analysis of tissue blocks asdescribed below.

[0089] If PMSR raised antisera are unable to detect the target proteinin whole cell extracts then the antibody will be affinity-purified bylinking the corresponding peptide to cyanogen bromide-activated agarosebeads following the manufacturer's protocol (Pierce). Total antiserumwill be incubated with affinity resin for 2 hours on a rotator wheel,washed in PBS buffer, followed by centrifugation for 5 cycles. Antibodyis liberated from resin by incubation in acidic glycine buffer. Freeantibody is added to neutralizing buffer in 1M Tris pH, 8.0. Antibody isthen re-tested as described above.

Example 3 Analysis Of Other Tumor Sources For PMSR2 And PMSR3 Expression

[0090] A preliminary analysis of PMSR2 and PMSR3 expression wasperformed using RNAs from primary tissues as well as on a subset ofcolorectal tumor tissues and cell lines. A more extensive survey ofother tissue types for PMSR2 and PMSR3 expression may be performed inlight of the wide distribution of MI tumors that lack detectablemutations in the previously identified MMR genes (Xu, L. et al. (2001)Int. J. Cancer 91:200-204). Samples may be tested using tissue panelspurchased from a supplier such as the NCI Tissue Array Research Program(TARP) sponsored by the Cooperative Human Tissue Network. Microarraysare screened with hPMSR2 and hPMSR3 antisera to monitor for expressionin neoplastic specimens.

[0091] Immunohistochemistry of slides are performed using a standardprotocol as described (Grasso, L. et al. (1998) J. Biol. Chem.273:24016-24024). Briefly, paraffin embedded sections are incubated inxylene for 10 minutes each, followed by 2 minutes incubation in 100%ethanol. Next, samples are hydrated by placing them in 95%, 70%, 50%,30% ethanol for 2 minutes each. Hydrated samples are then incubated for30 minutes in 0.3% hydrogen peroxide in methanol to block endogenousperoxidase activity. Slides are washed in a chamber of running water for20 minutes and placed in 0.25 M Tris-HCl pH 7.5 buffer. Forimmunostaining, slides are blocked with 10% goat serum in PBS for 20minutes at room temperature in a humidified chamber followed by a finalwash in PBS buffer. Antibody is diluted 1:20 in reaction buffercontaining 0.25 M Tris-HCl pH 7.5; 0.5% BSA and 2% fetal calf serum andadded onto the slide surface with enough volume to flood the tissuearea. Slides are incubated at room temperature for 4 hours and washed inPBS for 5 minutes, blocked in reaction buffer for 5 minutes and probedwith a secondary anti-rabbit HRP conjugated antibody diluted 1:200 inreaction buffer for 30 minutes in a humidified chamber. After secondarystaining, slides are washed for 5 minutes in buffer as before. Sectionsare visualized by peroxidase staining using the Vectastain kit(Amersham) following the manufacturer's instructions. Reactions arestopped by rinsing in water after a uniform brown color becomes visibleon the section. Reactions are carried out using antibodies with orwithout immunizing peptide as competitor to monitor for specificbinding. Slides are examined via microscopy and scored positive insamples where internal staining is observed when the appropriateantibody is incubated alone or in the presence of nonsense peptidecompetitor but negative when antibody is incubated with blockingpeptide. Samples will be repeated to confirm reproducibility.

Example 4 Generation Of Inducible MMR Dominant Negative Allele VectorsAnd Yeast Cells Harboring The Expression Vectors

[0092] Yeast expression constructs were prepared to determine if thehuman PMS2 related gene (hPMSR2) (Nicolaides, N. C. et al. (1995)Genomics 30(2):195-206) and the human PMS 134 gene (Nicolaides N. C. etal. (1998) Mol. Cell. Biol. 18:1635-1641) are capable of inactivatingthe yeast MMR activity and thereby increase the overall frequency ofgenomic hypermutation, a consequence of which is the generation ofvariant sib cells with novel output traits following host selection. Forthese studies, a plasmid encoding the hPMS 134 cDNA was altered bypolymerase chain reaction (PCR). The 5′ oligonucleotide has thefollowing structure: 5′-ACG CAT ATG GAG CGA GCT GAG AGC TCG AGT-3′ (SEQID NO:45) that includes the NdeI restriction site CAT ATG. The3′-oligonucleotide has the following structure: 5′-GAA TTC TTA TCA CGTAGA ATC GAG ACC GAG GAG AGG GTT AGG GAT AGG CTT ACC AGT TCC AAC CTT CGCCGA TGC-3′ (SEQ ID NO:46) that includes an EcoRI site GAA TTC and the 14amino acid epitope for the V5 antibody. The oligonucleotides were usedfor PCR under standard conditions that included 25 cycles of PCR (95° C.for 1 minute, 55° C. for 1 minute, 72° C. for 1.5 minutes for 25 cyclesfollowed by 3 minutes at 72° C.).

[0093] The PCR fragment was purified by gel electrophoresis and clonedinto pTA2.1 (Invitrogen) by standard cloning methods (Sambrook et al.MOLECULAR CLONING: A LABORATORY MANUAL, Third Edition, 2001), creatingthe plasmid pTA2.1-hPMS134. pTA2.1-hPMS 134 was digested with therestriction enzyme EcoRI to release the insert which was cloned intoEcoRI restriction site of pPIC3.5K (Invitrogen). The following strategy,similar to that described above to clone human PMS134, was used toconstruct an expression vector for the human related gene PMSR2. First,the hPMSR2 fragment was amplified by PCR to introduce two restrictionsites, an NdeI restriction site at the 5′end and an EcoRI site at the3′-end of the fragment. The 5′-oligonucleotide that was used for PCR hasthe following structure: 5′-ACG CAT ATG TGT CCT TGG CGG CCT AGA-3′ (SEQID NO:47) that includes the NdeI restriction site CAT ATG. The3′-oligonucleotide used for PCR has the following structure: 5′-GAA TTCTTA TTA CGT AGA ATC GAG ACC GAG GAG AGG GTT AGG GAT AGG CTT ACC CAT GTGTGA TGT TTC AGA GCT-3′ (SEQ ID NO:48) that includes an EcoRI site GAATTC and the V5 epitope to allow for antibody detection. The plasmid thatcontained human PMSR3 in pBluescript SK (Nicolaides N. C. et al. (1995)Genomics 30(2):195-206) was used as the PCR target with thehPMS2-specific oligonucleotides above. Following 25 cycles of PCR (95°C. for 1 minute, 55° C. for 1 minute, 72° C. for 1.5 minutes for 25cycles followed by 3 minutes at 72° C.). The PCR fragment was purifiedby gel electrophoresis and cloned into pTA2.1 (Invitrogen) by standardcloning methods (Sambrook et al., MOLECULAR CLONING: A LABORATORYMANUAL, Third Edition, 2001), creating the plasmid pTA2.1-hR2.pTA2.1-hR2 was next digested with the restriction enzyme EcoRI torelease the insert (there are two EcoRI restriction sites in themultiple cloning site of pTA2.1 that flank the insert) and the insertedinto the yeast expression vector pPIC3.5K (Invitrogen).

[0094]Pichia pastoris yeast cells were transformed with pPIC3.5K vector,pPIC3.5K-PMS134, and pPIC3.5K-hR2 as follows. First, 5 ml of YPD (1%yeast extract, 2% bacto-peptone, 1% dextrose) medium was inoculated witha single colony from a YPD plate (same as YPD liquid but add 2% Difcoagar to plate) and incubated with shaking overnight at 30° C. Theovernight culture was used to inoculate 500 ml of YPD medium (200 ul ofovernight culture) and the culture incubated at 30° C. until the opticaldensity at 600 nm reached 1.3 to 1.5. The cells were then spun down(4000×g for 10 minutes), and then washed 2 times in sterile water (onevolume each time), then the cells suspended in 20 ml of 1 M sorbitol.The sorbitol/cell suspension was spun down (4,000×g for 10 minutes) andsuspended in 1 ml of 1 M sorbitol. 80 ul of the cell suspension wasmixed with 5 to 10 ug of linearized plasmid DNA and placed in a 0.2 cmcuvette, pulsed length 5 to 10 milliseconds at field strength of 7,500V/cm. Next, the cells are diluted in 1 ml of 1 M sorbitol andtransferred to a 15 ml tube and incubated at 30° C. for 1 to 2 hourswithout shaking. Next, the cells are spun out (4,000×G for 10 minutes)and suspended in 100 ul of sterile water, and 50 ul/plate spread ontothe appropriate selective medium plate. The plates are incubated for 2to 3 days at 30° C. and colonies patched out onto YPD plates for furthertesting.

Example 5 Generation Of Hypermutable Yeast With Inducible DominantNegative Alleles Of Mismatch Repair Genes

[0095] Yeast clones expressing human PMS2 homologue PMS-R2 or emptyvector were grown in BMG (100 mM potassium phosphate, pH 6.0, 1.34% YNB(yeast nitrogen base), 4×10−5% biotin, 1% glycerol) liquid culture for24 hr at 30° C. The next day, cultures were diluted 1:100 in MM medium(1.34% YNB, 4×10−5% biotin, 0.5% methanol) and incubated at 30° C. withshaking. Cells were removed for mutant selection at 24 and 48 hours postmethanol induction as described below (see Example 6).

Example 6 Dominant Negative MMR Genes Can Produce New Genetic VariantsAnd Commercially Viable Output Traits In Yeast

[0096] The ability to express MMR genes in yeast, as presented inExample 5, demonstrates the ability to generate genetic alterations andnew phenotypes in yeast expressing dominant negative MMR genes. In thisexample we teach the utility of this method to create eukaryotic strainswith commercially relevant output traits.

Generation of Uracil Dependent Yeast Strain

[0097] One example of utility is the generation of a yeast strain thatis mutant for a particular metabolic product, such as an amino acid ornucleotide. Engineering such a yeast strain will allow for recombinantmanipulation of the yeast strain for the introduction of genes forscalable process of recombinant manufacturing. In order to demonstratethat MMR can be manipulated in yeast to generate mutants that lack theability to produce specific molecular building blocks, the followingexperiment was performed. Yeast cells that express a methanol induciblehuman PMS2 homologue, hPMS2-R2 (as described in Example 4 above), weregrown in BMY medium overnight then diluted 1:100 and transferred to MMmedium, which results in activation of the AOX promoter and productionof the hPMS2-R2 MMR gene that is resident within the yeast cell. Controlcells were treated the same manner; these cells contain the pPIC3.5vector in yeast and lack an insert. Cells were induced for 24 and 48hours and then selected for uracil requiring mutations as follows. Thecells were plated to 5-FOA medium (Boeke, J. D. et al. (1984) Mol. Gen.Genet. 197:345-346). The plates are made as follows: (2×concentrate(filter sterilize): yeast nitrogen base 7 grams; 5-fluoro-orotic acid 1gram; uracil 50 milligrams; glucose 20 grams; water to 500 ml; Add to500 ml 4% agar (autoclaved) and pour plates. Cells are plated on 5-FOAplates (0, 24 and 48 hour time points) and incubated at 30° C. forbetween 3 and 5 days. Data from a typical experiment is shown in Table3. No uracil requiring clones were observed in the un-induced or inducedculture in yeast cells that harbor the “empty” vector whereas thosecells that harbor the MMR gene hPMS2-R2 have clones that are capable ofgrowth on the selection medium. Note that the uninduced culture ofhPMS2-R2 does not have any colonies that are resistant to 5-FOA,demonstrating that the gene must be induced for the novel phenotype tobe generated.

[0098] It has been demonstrated that the mutagens (such as ethyl methylsulfonate result in a low number of ura mutants and that the spontaneousmutation rate for generating this class of mutants is low (Boeke, J. D.et al. (1984) Mol. Gen. Genet. 197:345-346). TABLE 3 Generation ofuracil requiring mutant Pichia pastoris yeast cells. Frequency StrainSeeded ura− URA+ (ura− cells) Wt 100,000 0 ˜100,000 0 Empty 100,000 0˜100,000 0 pMOR^(ye-1#) 100,000 14 ˜100,000 1/7,142 pMOR^(ye2@) 100,000123 ˜100,000 1/813 Wt 100,000 1-0.1 100,000 1/10^(5-6*) Mutagen 100,00010 100,000 1/10,000

Generation Of Heat-resistant Producer Strains

[0099] One example of commercial utility is the generation ofheat-resistant recombinant protein producer strains. In the scalableprocess of recombinant manufacturing, large-scale fermentation of bothprokaryotes and eukaryotes results in the generation of excessive heatwithin the culture. This heat must be dissipated by physical means suchas using cooling jackets that surround the culture while it is activelygrowing and producing product. Production of a yeast strain that canresist high temperature growth effectively would be advantageous forlarge-scale recombinant manufacturing processes. To this end, the yeaststrain as described in Example 5 can be grown in the presence ofmethanol to induce the dominant negative MMR gene and the cells grownfor various times (e.g. 12, 24, 36 and 48 hours) then put on plates andincubated at elevated temperatures to select for mutants that resisthigh temperature growth (e.g., 37° C. or 42° C.). These strains would beuseful for fermentation development and scale-up of processes and shouldresult in a decrease in manufacturing costs due to the need to cool thefermentation less often.

Generation Of High Recombinant Protein Producer Strains And Strains WithLess Endogenous Protease Activity

[0100] Yeast is a valuable recombinant-manufacturing organism since itis a single celled organism that is inexpensive to grow and easily lendsitself to fermentation at scale. Further more, many eukaryotic proteinsthat are incapable of folding effectively when expressed in Escherichiacoli systems fold with the proper conformation in yeast and arestructurally identical to their mammalian counterparts. There areseveral inherent limitations of many proteins that are expressed inyeast including over and/or inappropriate glycosylation of therecombinant protein, proteolysis by endogenous yeast enzymes andinsufficient secretion of recombinant protein from the inside of theyeast cell to the medium (which facilitates purification). To generateyeast cells that with this ability to over-secrete proteins, or withless endogenous protease activity and or less hyper-glycosylationactivity yeast cells as described in Example 4 can be grown withmethanol for 12, 24, 36 and 48 hours and yeast cells selected for theability to over-secrete the protein or interest, under-glycosylate it ora cell with attenuated of no protease activity. Such a strain will beuseful for recombinant manufacturing or other commercial purposes andcan be combined with the heat resistant strain outlined above.

[0101] For example, a mutant yeast cell that is resistant to hightemperature growth and can secrete large amounts of protein into themedium would result.

[0102] Similar results were observed with other dominant negativemutants such as the PMSR2, PMSR3, and the human MLH1 proteins.

Example 7 Mutations Generated In The Host Genome Of Yeast By DefectiveMMR Are Genetically Stable

[0103] As described in Example 6 manipulation of the MMR pathway inyeast results in alterations within the host genome and the ability toselect for a novel output traits, for example the ability of a yeastcell to require a specific nutrient. It is important that the mutationsintroduced by the MMR pathway is genetically stable and passed todaughter cells reproducibly once the wild type MMR pathway isre-established. To determine the genetic stability of mutationsintroduced into the yeast genome the following experiment was performed.Five independent colonies from pPIC3.5KhPMS2-R2 that are ura-, five wildtype control cells (URA+) and five pPIC3.5K transformed cells (“emptyvector”) were grown overnight from an isolated colony in 5 ml of YPD (1%yeast extract, 2% bacto-peptone and 1% dextrose) at 30° C. with shaking.The YPD medium contains all the nutrients necessary for yeast to grow,including uracil. Next, 1 pL of the overnight culture, which was at anoptical density (OD) as measured at 600 nM of >3.0, was diluted to anOD600 of 0.01 in YPD and the culture incubated with shaking at 30° C.for an additional 24 hours. This process was repeated 3 more times for atotal of 5 overnight incubations. This is the equivalent of greater than100 generations of doubling (from the initial colony on the plate to theend of the last overnight incubation. Cells (five independent coloniesthat are ura and five that were wild type were then plated onto YPDplates at a cell density of 300 to 1,000 cells/plate and incubated fortwo days at 30° C. The cells from these plates were replica plated tothe following plates and scored for growth following three daysincubation at 30° C.; Synthetic Complete (SC) SC-ura (1.34% yeastnitrogen base and ammonium sulfate; 4×10−5% biotin; supplemented withall amino acids, NO supplemental uracil; 2% dextrose and 2% agar); SC+URA (same as SC-ura but supplement plate with 50 mg uracil/litermedium), and YPD plates. They were replica plated in the followingorder-SC-ura, SC complete, YPD. If the novel output trait that isresident within the yeast genome that was generated by expression of themutant MMR (in this example the human homologue of PMS2, hPMS2-R2) isunstable, the uracil dependent cells should “revert” back a uracilindependent phenotype. If the phenotype is stable, growth of the mutantcells under non-selective conditions should result in yeast cells thatmaintain their viability dependence on exogenous supplementation withuracil. As can be seen in the data presented in Table 4, the uracildependent phenotype is stable when the yeast cells are grown undernon-selective conditions, demonstrating that the MMR-generated phenotypederived from mutation in one of the uracil biosynthetic pathway genes isstable genetically. TABLE 4 Strain Seeded −ura +URA YPD Wt 650 650 650650 Empty 560 560 560 560 pMOR^(ye-1#) 730 0 730 730

[0104] These data demonstrate the utility of employing an inducibleexpression system and a dominant negative MMR gene in a eukaryoticsystem to generate genetically altered strains. The strain developed inthis example, a yeast strain that now requires addition of uracil forgrowth, is potentially useful as a strain for recombinant manufacturing;by constructing an expression vector that harbors the wild type URA3gene on either an integration plasmid or an extra-chromosomal vector itis now possible to transform and create novel cells expressing the aprotein of interest. It is also possible to modify other resident genesin yeast cells and select for mutations in genes that that give otheruseful phenotypes, such as the ability to carry out a novelbiotransformation. Furthermore, it is possible to express a geneextra-chromosomally in a yeast cell that has altered MMR activity asdescribed above and select for mutations in the extra-chromosomal gene.Therefore, in a similar manner to that described above the mutant yeastcell can be put under specific selective pressure and a novel proteinwith commercially important biochemical attributes selected.

[0105] These examples are meant only as illustrations and are not meantto limit the scope of the present invention.

[0106] Finally, as described above once a mutation has been introducedinto the gene of interest the MMR activity is attenuated of completelyabolished. The result is a yeast cell that harbors a stable mutation inthe target gene(s) of interest.

Example 8 Enhanced Generation Of MMR-Defective Yeast And ChemicalMutagens For The Generation Of New Output Traits

[0107] It has been previously documented that MMR deficiency yields toincreased mutation frequency and increased resistance to toxic effectsof chemical mutagens (CM) and their respective analogues such as but notlimited to those as: ethidium bromide, EMS, MNNG, MNU, Tamoxifen,8-Hydroxyguanine, as well as others listed but not limited to inpublications by: Khromov-Borisov, N. N., et al. (1999) Mutat. Res.430:55-74; Ohe, T. et al. (1999) Mutat. Res. 429:189-199; Hour, T. C. etal. (1999) Food Chem. Toxicol. 37:569-579; Hrelia, P. et al. (1999)Chem. Biol. Interact. 118:99-111; Garganta, F. et al. (1999) Environ.Mol. Mutagen. 33:75-85; Ukawa-Ishikawa S. et al. (1998) Mutat. Res.412:99-107; www.ehs.utah.edu/ohh/mutagens; Marcelino, L. A. et al (1998)Cancer Res. 58(13):2857-2862; Koi, M. et al. (1994) Cancer Res.54:4308-4312. Mismatch repair provokes chromosome aberrations in hamstercells treated with methylating agents or 6thioguanine, but not withethylating agents. To demonstrate the ability of CMs to increase themutation frequency in MMR defective yeast cells, we would predict thatexposure of yeast cells to CMs in the presence or absence of methanol(which induces the expression of the resident human homologue to PMS2,hPMS2-R2) will result in an augmentation of mutations within the yeastcell.

[0108] Yeast cells that express hPMS2-R2 (induced or un-induced) andempty vector control cells are grown as described in Examples 5 and 6)and for 24 hours and diluted into MM medium as described above. Next,the cells in MM are incubated either with or without increasing amountsof ethyl methane sulfonate (EMS) from 0, 1, 10, 50, 100, and 200 pM. 10zip aliquots of culture (diluted in 300 ul MM) and incubated for 30minutes, 60 minutes, and 120 minutes followed by plating cells onto5-FOA plates as described in Example 3 above. Mutants are selected andscored as above. We would predict that there will be an increase in thefrequency of ura mutants in the PMS2-R2 cultures that are induced withmethanol as compared to the uninduced parental or wild type strain. In afurther extension of this example, human PMS2-R2 harboring cells will beinduced for 24 and 48 hours then mutagenized with EMS. This will allowthe MMR gene to be fully active and expressed at high levels, therebyresulting in an increase in the number of ura mutants obtained. We wouldpredict that there will be no change in the number of ura mutantsobtained in the uninduced parental control or the wild type “emptyvector” cells. This example demonstrates the use of employing aregulated dominant negative MMR system plus chemical mutagens to produceenhanced numbers of genetically altered yeast strains that can beselected for new output traits. This method is useful for generatingsuch organisms for commercial applications such as but not limited torecombinant manufacturing, biotransformation, and altered biochemicalswith enhanced activities. It is also useful to obtain alterations ofprotein activity from ectopically expressed proteins harbored onextra-chromosomal expression vectors similar to those described inExample 4 above.

Examples Of MMR Genes And Encoded Polypeptides

[0109] Yeast MLH1 cDNA (accession number U07187) (SEQ ID NO:1); yeastMLH1 protein (accession number U07187) (SEQ ID NO:2); mouse PMS2 protein(SEQ ID NO:3); mouse PMS2 cDNA (SEQ ID NO:4); human PMS2 protein (SEQ IDNO:5); human PMS2 cDNA (SEQ ID NO:6); human PMS1 protein (SEQ ID NO:7);human PMS1 cDNA (SEQ ID NO:8); human MSH2 protein (SEQ ID NO:9); humanMSH2 cDNA (SEQ ID NO:10); human MLHI protein (SEQ ID NO:11); human MLH1cDNA (SEQ ID NO:12); hPMS2-134 protein (SEQ ID NO:13); hPMS2-134 cDNA(SEQ ID NO:14); hMSH6 (human protein) (accession number U28946 (SEQ IDNO:15); hMSH6 (human cDNA) (accession number U28946) (SEQ ID NO:16);hPMSR2 (human cDNA) (accession number U38964) (SEQ ID NO:17); hPMSR2(human protein) (accession number U38964) (SEQ ID NO:18); HPMSR3 (humancDNA) (accession number NM_(—)005395.1) (SEQ ID NO:19); hPMSR3 (humanprotein) (accession number U38979.1) (SEQ ID NO:20); hPMSR6 (human cDNA)(accession number U38980.1) (SEQ ID NO:21); hPMSR6 (human protein)(accession number U38980.1) (SEQ ID NO:22).

1 48 1 3218 DNA Saccharomyces cerevisiae 1 aaataggaat gtgataccttctattgcatg caaagatagt gtaggaggcg ctgctattgc 60 caaagacttt tgagaccgcttgctgtttca ttatagttga ggagttctcg aagacgagaa 120 attagcagtt ttcggtgtttagtaatcgcg ctagcatgct aggacaattt aactgcaaaa 180 ttttgatacg atagtgatagtaaatggaag gtaaaaataa catagaccta tcaataagca 240 atgtctctca gaataaaagcacttgatgca tcagtggtta acaaaattgc tgcaggtgag 300 atcataatat cccccgtaaatgctctcaaa gaaatgatgg agaattccat cgatgcgaat 360 gctacaatga ttgatattctagtcaaggaa ggaggaatta aggtacttca aataacagat 420 aacggatctg gaattaataaagcagacctg ccaatcttat gtgagcgatt cacgacgtcc 480 aaattacaaa aattcgaagatttgagtcag attcaaacgt atggattccg aggagaagct 540 ttagccagta tctcacatgtggcaagagtc acagtaacga caaaagttaa agaagacaga 600 tgtgcatgga gagtttcatatgcagaaggt aagatgttgg aaagccccaa acctgttgct 660 ggaaaagacg gtaccacgatcctagttgaa gacctttttt tcaatattcc ttctagatta 720 agggccttga ggtcccataatgatgaatac tctaaaatat tagatgttgt cgggcgatac 780 gccattcatt ccaaggacattggcttttct tgtaaaaagt tcggagactc taattattct 840 ttatcagtta aaccttcatatacagtccag gataggatta ggactgtgtt caataaatct 900 gtggcttcga atttaattacttttcatatc agcaaagtag aagatttaaa cctggaaagc 960 gttgatggaa aggtgtgtaatttgaatttc atatccaaaa agtccatttc attaattttt 1020 ttcattaata atagactagtgacatgtgat cttctaagaa gagctttgaa cagcgtttac 1080 tccaattatc tgccaaagggcttcagacct tttatttatt tgggaattgt tatagatccg 1140 gcggctgttg atgttaacgttcacccgaca aagagagagg ttcgtttcct gagccaagat 1200 gagatcatag agaaaatcgccaatcaattg cacgccgaat tatctgccat tgatacttca 1260 cgtactttca aggcttcttcaatttcaaca aacaagccag agtcattgat accatttaat 1320 gacaccatag aaagtgataggaataggaag agtctccgac aagcccaagt ggtagagaat 1380 tcatatacga cagccaatagtcaactaagg aaagcgaaaa gacaagagaa taaactagtc 1440 agaatagatg cttcacaagctaaaattacg tcatttttat cctcaagtca acagttcaac 1500 tttgaaggat cgtctacaaagcgacaactg agtgaaccca aggtaacaaa tgtaagccac 1560 tcccaagagg cagaaaagctgacactaaat gaaagcgaac aaccgcgtga tgccaataca 1620 atcaatgata atgacttgaaggatcaacct aagaagaaac aaaagttggg ggattataaa 1680 gttccaagca ttgccgatgacgaaaagaat gcactcccga tttcaaaaga cgggtatatt 1740 agagtaccta aggagcgagttaatgttaat cttacgagta tcaagaaatt gcgtgaaaaa 1800 gtagatgatt cgatacatcgagaactaaca gacatttttg caaatttgaa ttacgttggg 1860 gttgtagatg aggaaagaagattagccgct attcagcatg acttaaagct ttttttaata 1920 gattacggat ctgtgtgctatgagctattc tatcagattg gtttgacaga cttcgcaaac 1980 tttggtaaga taaacctacagagtacaaat gtgtcagatg atatagtttt gtataatctc 2040 ctatcagaat ttgacgagttaaatgacgat gcttccaaag aaaaaataat tagtaaaata 2100 tgggacatga gcagtatgctaaatgagtac tattccatag aattggtgaa tgatggtcta 2160 gataatgact taaagtctgtgaagctaaaa tctctaccac tacttttaaa aggctacatt 2220 ccatctctgg tcaagttaccattttttata tatcgcctgg gtaaagaagt tgattgggag 2280 gatgaacaag agtgtctagatggtatttta agagagattg cattactcta tatacctgat 2340 atggttccga aagtcgatacactcgatgca tcgttgtcag aagacgaaaa agcccagttt 2400 ataaatagaa aggaacacatatcctcatta ctagaacacg ttctcttccc ttgtatcaaa 2460 cgaaggttcc tggcccctagacacattctc aaggatgtcg tggaaatagc caaccttcca 2520 gatctataca aagtttttgagaggtgttaa ctttaaaacg ttttggctgt aataccaaag 2580 tttttgttta tttcctgagtgtgattgtgt ttcatttgaa agtgtatgcc ctttccttta 2640 acgattcatc cgcgagatttcaaaggatat gaaatatggt tgcagttagg aaagtatgtc 2700 agaaatgtat attcggattgaaactcttct aatagttctg aagtcacttg gttccgtatt 2760 gttttcgtcc tcttcctcaagcaacgattc ttgtctaagc ttattcaacg gtaccaaaga 2820 cccgagtcct tttatgagagaaaacatttc atcatttttc aactcaatta tcttaatatc 2880 attttgtagt attttgaaaacaggatggta aaacgaatca cctgaatcta gaagctgtac 2940 cttgtcccat aaaagttttaatttactgag cctttcggtc aagtaaacta gtttatctag 3000 ttttgaaccg aatattgtgggcagatttgc agtaagttca gttagatcta ctaaaagttg 3060 tttgacagca gccgattccacaaaaatttg gtaaaaggag atgaaagaga cctcgcgcgt 3120 aatggtttgc atcaccatcggatgtctgtt gaaaaactca ctttttgcat ggaagttatt 3180 aacaataaga ctaatgattaccttagaata atgtataa 3218 2 769 PRT Saccharomyces cerevisiae 2 Met SerLeu Arg Ile Lys Ala Leu Asp Ala Ser Val Val Asn Lys Ile 1 5 10 15 AlaAla Gly Glu Ile Ile Ile Ser Pro Val Asn Ala Leu Lys Glu Met 20 25 30 MetGlu Asn Ser Ile Asp Ala Asn Ala Thr Met Ile Asp Ile Leu Val 35 40 45 LysGlu Gly Gly Ile Lys Val Leu Gln Ile Thr Asp Asn Gly Ser Gly 50 55 60 IleAsn Lys Ala Asp Leu Pro Ile Leu Cys Glu Arg Phe Thr Thr Ser 65 70 75 80Lys Leu Gln Lys Phe Glu Asp Leu Ser Gln Ile Gln Thr Tyr Gly Phe 85 90 95Arg Gly Glu Ala Leu Ala Ser Ile Ser His Val Ala Arg Val Thr Val 100 105110 Thr Thr Lys Val Lys Glu Asp Arg Cys Ala Trp Arg Val Ser Tyr Ala 115120 125 Glu Gly Lys Met Leu Glu Ser Pro Lys Pro Val Ala Gly Lys Asp Gly130 135 140 Thr Thr Ile Leu Val Glu Asp Leu Phe Phe Asn Ile Pro Ser ArgLeu 145 150 155 160 Arg Ala Leu Arg Ser His Asn Asp Glu Tyr Ser Lys IleLeu Asp Val 165 170 175 Val Gly Arg Tyr Ala Ile His Ser Lys Asp Ile GlyPhe Ser Cys Lys 180 185 190 Lys Phe Gly Asp Ser Asn Tyr Ser Leu Ser ValLys Pro Ser Tyr Thr 195 200 205 Val Gln Asp Arg Ile Arg Thr Val Phe AsnLys Ser Val Ala Ser Asn 210 215 220 Leu Ile Thr Phe His Ile Ser Lys ValGlu Asp Leu Asn Leu Glu Ser 225 230 235 240 Val Asp Gly Lys Val Cys AsnLeu Asn Phe Ile Ser Lys Lys Ser Ile 245 250 255 Ser Leu Ile Phe Phe IleAsn Asn Arg Leu Val Thr Cys Asp Leu Leu 260 265 270 Arg Arg Ala Leu AsnSer Val Tyr Ser Asn Tyr Leu Pro Lys Gly Phe 275 280 285 Arg Pro Phe IleTyr Leu Gly Ile Val Ile Asp Pro Ala Ala Val Asp 290 295 300 Val Asn ValHis Pro Thr Lys Arg Glu Val Arg Phe Leu Ser Gln Asp 305 310 315 320 GluIle Ile Glu Lys Ile Ala Asn Gln Leu His Ala Glu Leu Ser Ala 325 330 335Ile Asp Thr Ser Arg Thr Phe Lys Ala Ser Ser Ile Ser Thr Asn Lys 340 345350 Pro Glu Ser Leu Ile Pro Phe Asn Asp Thr Ile Glu Ser Asp Arg Asn 355360 365 Arg Lys Ser Leu Arg Gln Ala Gln Val Val Glu Asn Ser Tyr Thr Thr370 375 380 Ala Asn Ser Gln Leu Arg Lys Ala Lys Arg Gln Glu Asn Lys LeuVal 385 390 395 400 Arg Ile Asp Ala Ser Gln Ala Lys Ile Thr Ser Phe LeuSer Ser Ser 405 410 415 Gln Gln Phe Asn Phe Glu Gly Ser Ser Thr Lys ArgGln Leu Ser Glu 420 425 430 Pro Lys Val Thr Asn Val Ser His Ser Gln GluAla Glu Lys Leu Thr 435 440 445 Leu Asn Glu Ser Glu Gln Pro Arg Asp AlaAsn Thr Ile Asn Asp Asn 450 455 460 Asp Leu Lys Asp Gln Pro Lys Lys LysGln Lys Leu Gly Asp Tyr Lys 465 470 475 480 Val Pro Ser Ile Ala Asp AspGlu Lys Asn Ala Leu Pro Ile Ser Lys 485 490 495 Asp Gly Tyr Ile Arg ValPro Lys Glu Arg Val Asn Val Asn Leu Thr 500 505 510 Ser Ile Lys Lys LeuArg Glu Lys Val Asp Asp Ser Ile His Arg Glu 515 520 525 Leu Thr Asp IlePhe Ala Asn Leu Asn Tyr Val Gly Val Val Asp Glu 530 535 540 Glu Arg ArgLeu Ala Ala Ile Gln His Asp Leu Lys Leu Phe Leu Ile 545 550 555 560 AspTyr Gly Ser Val Cys Tyr Glu Leu Phe Tyr Gln Ile Gly Leu Thr 565 570 575Asp Phe Ala Asn Phe Gly Lys Ile Asn Leu Gln Ser Thr Asn Val Ser 580 585590 Asp Asp Ile Val Leu Tyr Asn Leu Leu Ser Glu Phe Asp Glu Leu Asn 595600 605 Asp Asp Ala Ser Lys Glu Lys Ile Ile Ser Lys Ile Trp Asp Met Ser610 615 620 Ser Met Leu Asn Glu Tyr Tyr Ser Ile Glu Leu Val Asn Asp GlyLeu 625 630 635 640 Asp Asn Asp Leu Lys Ser Val Lys Leu Lys Ser Leu ProLeu Leu Leu 645 650 655 Lys Gly Tyr Ile Pro Ser Leu Val Lys Leu Pro PhePhe Ile Tyr Arg 660 665 670 Leu Gly Lys Glu Val Asp Trp Glu Asp Glu GlnGlu Cys Leu Asp Gly 675 680 685 Ile Leu Arg Glu Ile Ala Leu Leu Tyr IlePro Asp Met Val Pro Lys 690 695 700 Val Asp Thr Leu Asp Ala Ser Leu SerGlu Asp Glu Lys Ala Gln Phe 705 710 715 720 Ile Asn Arg Lys Glu His IleSer Ser Leu Leu Glu His Val Leu Phe 725 730 735 Pro Cys Ile Lys Arg ArgPhe Leu Ala Pro Arg His Ile Leu Lys Asp 740 745 750 Val Val Glu Ile AlaAsn Leu Pro Asp Leu Tyr Lys Val Phe Glu Arg 755 760 765 Cys 3 859 PRTMus musculus 3 Met Glu Gln Thr Glu Gly Val Ser Thr Glu Cys Ala Lys AlaIle Lys 1 5 10 15 Pro Ile Asp Gly Lys Ser Val His Gln Ile Cys Ser GlyGln Val Ile 20 25 30 Leu Ser Leu Ser Thr Ala Val Lys Glu Leu Ile Glu AsnSer Val Asp 35 40 45 Ala Gly Ala Thr Thr Ile Asp Leu Arg Leu Lys Asp TyrGly Val Asp 50 55 60 Leu Ile Glu Val Ser Asp Asn Gly Cys Gly Val Glu GluGlu Asn Phe 65 70 75 80 Glu Gly Leu Ala Leu Lys His His Thr Ser Lys IleGln Glu Phe Ala 85 90 95 Asp Leu Thr Gln Val Glu Thr Phe Gly Phe Arg GlyGlu Ala Leu Ser 100 105 110 Ser Leu Cys Ala Leu Ser Asp Val Thr Ile SerThr Cys His Gly Ser 115 120 125 Ala Ser Val Gly Thr Arg Leu Val Phe AspHis Asn Gly Lys Ile Thr 130 135 140 Gln Lys Thr Pro Tyr Pro Arg Pro LysGly Thr Thr Val Ser Val Gln 145 150 155 160 His Leu Phe Tyr Thr Leu ProVal Arg Tyr Lys Glu Phe Gln Arg Asn 165 170 175 Ile Lys Lys Glu Tyr SerLys Met Val Gln Val Leu Gln Ala Tyr Cys 180 185 190 Ile Ile Ser Ala GlyVal Arg Val Ser Cys Thr Asn Gln Leu Gly Gln 195 200 205 Gly Lys Arg HisAla Val Val Cys Thr Ser Gly Thr Ser Gly Met Lys 210 215 220 Glu Asn IleGly Ser Val Phe Gly Gln Lys Gln Leu Gln Ser Leu Ile 225 230 235 240 ProPhe Val Gln Leu Pro Pro Ser Asp Ala Val Cys Glu Glu Tyr Gly 245 250 255Leu Ser Thr Ser Gly Arg His Lys Thr Phe Ser Thr Phe Arg Ala Ser 260 265270 Phe His Ser Ala Arg Thr Ala Pro Gly Gly Val Gln Gln Thr Gly Ser 275280 285 Phe Ser Ser Ser Ile Arg Gly Pro Val Thr Gln Gln Arg Ser Leu Ser290 295 300 Leu Ser Met Arg Phe Tyr His Met Tyr Asn Arg His Gln Tyr ProPhe 305 310 315 320 Val Val Leu Asn Val Ser Val Asp Ser Glu Cys Val AspIle Asn Val 325 330 335 Thr Pro Asp Lys Arg Gln Ile Leu Leu Gln Glu GluLys Leu Leu Leu 340 345 350 Ala Val Leu Lys Thr Ser Leu Ile Gly Met PheAsp Ser Asp Ala Asn 355 360 365 Lys Leu Asn Val Asn Gln Gln Pro Leu LeuAsp Val Glu Gly Asn Leu 370 375 380 Val Lys Leu His Thr Ala Glu Leu GluLys Pro Val Pro Gly Lys Gln 385 390 395 400 Asp Asn Ser Pro Ser Leu LysSer Thr Ala Asp Glu Lys Arg Val Ala 405 410 415 Ser Ile Ser Arg Leu ArgGlu Ala Phe Ser Leu His Pro Thr Lys Glu 420 425 430 Ile Lys Ser Arg GlyPro Glu Thr Ala Glu Leu Thr Arg Ser Phe Pro 435 440 445 Ser Glu Lys ArgGly Val Leu Ser Ser Tyr Pro Ser Asp Val Ile Ser 450 455 460 Tyr Arg GlyLeu Arg Gly Ser Gln Asp Lys Leu Val Ser Pro Thr Asp 465 470 475 480 SerPro Gly Asp Cys Met Asp Arg Glu Lys Ile Glu Lys Asp Ser Gly 485 490 495Leu Ser Ser Thr Ser Ala Gly Ser Glu Glu Glu Phe Ser Thr Pro Glu 500 505510 Val Ala Ser Ser Phe Ser Ser Asp Tyr Asn Val Ser Ser Leu Glu Asp 515520 525 Arg Pro Ser Gln Glu Thr Ile Asn Cys Gly Asp Leu Asp Cys Arg Pro530 535 540 Pro Gly Thr Gly Gln Ser Leu Lys Pro Glu Asp His Gly Tyr GlnCys 545 550 555 560 Lys Ala Leu Pro Leu Ala Arg Leu Ser Pro Thr Asn AlaLys Arg Phe 565 570 575 Lys Thr Glu Glu Arg Pro Ser Asn Val Asn Ile SerGln Arg Leu Pro 580 585 590 Gly Pro Gln Ser Thr Ser Ala Ala Glu Val AspVal Ala Ile Lys Met 595 600 605 Asn Lys Arg Ile Val Leu Leu Glu Phe SerLeu Ser Ser Leu Ala Lys 610 615 620 Arg Met Lys Gln Leu Gln His Leu LysAla Gln Asn Lys His Glu Leu 625 630 635 640 Ser Tyr Arg Lys Phe Arg AlaLys Ile Cys Pro Gly Glu Asn Gln Ala 645 650 655 Ala Glu Asp Glu Leu ArgLys Glu Ile Ser Lys Ser Met Phe Ala Glu 660 665 670 Met Glu Ile Leu GlyGln Phe Asn Leu Gly Phe Ile Val Thr Lys Leu 675 680 685 Lys Glu Asp LeuPhe Leu Val Asp Gln His Ala Ala Asp Glu Lys Tyr 690 695 700 Asn Phe GluMet Leu Gln Gln His Thr Val Leu Gln Ala Gln Arg Leu 705 710 715 720 IleThr Pro Gln Thr Leu Asn Leu Thr Ala Val Asn Glu Ala Val Leu 725 730 735Ile Glu Asn Leu Glu Ile Phe Arg Lys Asn Gly Phe Asp Phe Val Ile 740 745750 Asp Glu Asp Ala Pro Val Thr Glu Arg Ala Lys Leu Ile Ser Leu Pro 755760 765 Thr Ser Lys Asn Trp Thr Phe Gly Pro Gln Asp Ile Asp Glu Leu Ile770 775 780 Phe Met Leu Ser Asp Ser Pro Gly Val Met Cys Arg Pro Ser ArgVal 785 790 795 800 Arg Gln Met Phe Ala Ser Arg Ala Cys Arg Lys Ser ValMet Ile Gly 805 810 815 Thr Ala Leu Asn Ala Ser Glu Met Lys Lys Leu IleThr His Met Gly 820 825 830 Glu Met Asp His Pro Trp Asn Cys Pro His GlyArg Pro Thr Met Arg 835 840 845 His Val Ala Asn Leu Asp Val Ile Ser GlnAsn 850 855 4 3056 DNA Mus musculus 4 gaattccggt gaaggtcctg aagaatttccagattcctga gtatcattgg aggagacaga 60 taacctgtcg tcaggtaacg atggtgtatatgcaacagaa atgggtgttc ctggagacgc 120 gtcttttccc gagagcggca ccgcaactctcccgcggtga ctgtgactgg aggagtcctg 180 catccatgga gcaaaccgaa ggcgtgagtacagaatgtgc taaggccatc aagcctattg 240 atgggaagtc agtccatcaa atttgttctgggcaggtgat actcagttta agcaccgctg 300 tgaaggagtt gatagaaaat agtgtagatgctggtgctac tactattgat ctaaggctta 360 aagactatgg ggtggacctc attgaagtttcagacaatgg atgtggggta gaagaagaaa 420 actttgaagg tctagctctg aaacatcacacatctaagat tcaagagttt gccgacctca 480 cgcaggttga aactttcggc tttcggggggaagctctgag ctctctgtgt gcactaagtg 540 atgtcactat atctacctgc cacgggtctgcaagcgttgg gactcgactg gtgtttgacc 600 ataatgggaa aatcacccag aaaactccctacccccgacc taaaggaacc acagtcagtg 660 tgcagcactt attttataca ctacccgtgcgttacaaaga gtttcagagg aacattaaaa 720 aggagtattc caaaatggtg caggtcttacaggcgtactg tatcatctca gcaggcgtcc 780 gtgtaagctg cactaatcag ctcggacaggggaagcggca cgctgtggtg tgcacaagcg 840 gcacgtctgg catgaaggaa aatatcgggtctgtgtttgg ccagaagcag ttgcaaagcc 900 tcattccttt tgttcagctg ccccctagtgacgctgtgtg tgaagagtac ggcctgagca 960 cttcaggacg ccacaaaacc ttttctacgtttcgggcttc atttcacagt gcacgcacgg 1020 cgccgggagg agtgcaacag acaggcagtttttcttcatc aatcagaggc cctgtgaccc 1080 agcaaaggtc tctaagcttg tcaatgaggttttatcacat gtataaccgg catcagtacc 1140 catttgtcgt ccttaacgtt tccgttgactcagaatgtgt ggatattaat gtaactccag 1200 ataaaaggca aattctacta caagaagagaagctattgct ggccgtttta aagacctcct 1260 tgataggaat gtttgacagt gatgcaaacaagcttaatgt caaccagcag ccactgctag 1320 atgttgaagg taacttagta aagctgcatactgcagaact agaaaagcct gtgccaggaa 1380 agcaagataa ctctccttca ctgaagagcacagcagacga gaaaagggta gcatccatct 1440 ccaggctgag agaggccttt tctcttcatcctactaaaga gatcaagtct aggggtccag 1500 agactgctga actgacacgg agttttccaagtgagaaaag gggcgtgtta tcctcttatc 1560 cttcagacgt catctcttac agaggcctccgtggctcgca ggacaaattg gtgagtccca 1620 cggacagccc tggtgactgt atggacagagagaaaataga aaaagactca gggctcagca 1680 gcacctcagc tggctctgag gaagagttcagcaccccaga agtggccagt agctttagca 1740 gtgactataa cgtgagctcc ctagaagacagaccttctca ggaaaccata aactgtggtg 1800 acctggactg ccgtcctcca ggtacaggacagtccttgaa gccagaagac catggatatc 1860 aatgcaaagc tctacctcta gctcgtctgtcacccacaaa tgccaagcgc ttcaagacag 1920 aggaaagacc ctcaaatgtc aacatttctcaaagattgcc tggtcctcag agcacctcag 1980 cagctgaggt cgatgtagcc ataaaaatgaataagagaat cgtgctcctc gagttctctc 2040 tgagttctct agctaagcga atgaagcagttacagcacct aaaggcgcag aacaaacatg 2100 aactgagtta cagaaaattt agggccaagatttgccctgg agaaaaccaa gcagcagaag 2160 atgaactcag aaaagagatt agtaaatcgatgtttgcaga gatggagatc ttgggtcagt 2220 ttaacctggg atttatagta accaaactgaaagaggacct cttcctggtg gaccagcatg 2280 ctgcggatga gaagtacaac tttgagatgctgcagcagca cacggtgctc caggcgcaga 2340 ggctcatcac accccagact ctgaacttaactgctgtcaa tgaagctgta ctgatagaaa 2400 atctggaaat attcagaaag aatggctttgactttgtcat tgatgaggat gctccagtca 2460 ctgaaagggc taaattgatt tccttaccaactagtaaaaa ctggaccttt ggaccccaag 2520 atatagatga actgatcttt atgttaagtgacagccctgg ggtcatgtgc cggccctcac 2580 gagtcagaca gatgtttgct tccagagcctgtcggaagtc agtgatgatt ggaacggcgc 2640 tcaatgcgag cgagatgaag aagctcatcacccacatggg tgagatggac cacccctgga 2700 actgccccca cggcaggcca accatgaggcacgttgccaa tctggatgtc atctctcaga 2760 actgacacac cccttgtagc atagagtttattacagattg ttcggtttgc aaagagaagg 2820 ttttaagtaa tctgattatc gttgtacaaaaattagcatg ctgctttaat gtactggatc 2880 catttaaaag cagtgttaag gcaggcatgatggagtgttc ctctagctca gctacttggg 2940 tgatccggtg ggagctcatg tgagcccaggactttgagac cactccgagc cacattcatg 3000 agactcaatt caaggacaaa aaaaaaaagatatttttgaa gccttttaaa aaaaaa 3056 5 932 PRT Homo sapiens 5 Met Lys GlnLeu Pro Ala Ala Thr Val Arg Leu Leu Ser Ser Ser Gln 1 5 10 15 Ile IleThr Ser Val Val Ser Val Val Lys Glu Leu Ile Glu Asn Ser 20 25 30 Leu AspAla Gly Ala Thr Ser Val Asp Val Lys Leu Glu Asn Tyr Gly 35 40 45 Phe AspLys Ile Glu Val Arg Asp Asn Gly Glu Gly Ile Lys Ala Val 50 55 60 Asp AlaPro Val Met Ala Met Lys Tyr Tyr Thr Ser Lys Ile Asn Ser 65 70 75 80 HisGlu Asp Leu Glu Asn Leu Thr Thr Tyr Gly Phe Arg Gly Glu Ala 85 90 95 LeuGly Ser Ile Cys Cys Ile Ala Glu Val Leu Ile Thr Thr Arg Thr 100 105 110Ala Ala Asp Asn Phe Ser Thr Gln Tyr Val Leu Asp Gly Ser Gly His 115 120125 Ile Leu Ser Gln Lys Pro Ser His Leu Gly Gln Gly Thr Thr Val Thr 130135 140 Ala Leu Arg Leu Phe Lys Asn Leu Pro Val Arg Lys Gln Phe Tyr Ser145 150 155 160 Thr Ala Lys Lys Cys Lys Asp Glu Ile Lys Lys Ile Gln AspLeu Leu 165 170 175 Met Ser Phe Gly Ile Leu Lys Pro Asp Leu Arg Ile ValPhe Val His 180 185 190 Asn Lys Ala Val Ile Trp Gln Lys Ser Arg Val SerAsp His Lys Met 195 200 205 Ala Leu Met Ser Val Leu Gly Thr Ala Val MetAsn Asn Met Glu Ser 210 215 220 Phe Gln Tyr His Ser Glu Glu Ser Gln IleTyr Leu Ser Gly Phe Leu 225 230 235 240 Pro Lys Cys Asp Ala Asp His SerPhe Thr Ser Leu Ser Thr Pro Glu 245 250 255 Arg Ser Phe Ile Phe Ile AsnSer Arg Pro Val His Gln Lys Asp Ile 260 265 270 Leu Lys Leu Ile Arg HisHis Tyr Asn Leu Lys Cys Leu Lys Glu Ser 275 280 285 Thr Arg Leu Tyr ProVal Phe Phe Leu Lys Ile Asp Val Pro Thr Ala 290 295 300 Asp Val Asp ValAsn Leu Thr Pro Asp Lys Ser Gln Val Leu Leu Gln 305 310 315 320 Asn LysGlu Ser Val Leu Ile Ala Leu Glu Asn Leu Met Thr Thr Cys 325 330 335 TyrGly Pro Leu Pro Ser Thr Asn Ser Tyr Glu Asn Asn Lys Thr Asp 340 345 350Val Ser Ala Ala Asp Ile Val Leu Ser Lys Thr Ala Glu Thr Asp Val 355 360365 Leu Phe Asn Lys Val Glu Ser Ser Gly Lys Asn Tyr Ser Asn Val Asp 370375 380 Thr Ser Val Ile Pro Phe Gln Asn Asp Met His Asn Asp Glu Ser Gly385 390 395 400 Lys Asn Thr Asp Asp Cys Leu Asn His Gln Ile Ser Ile GlyAsp Phe 405 410 415 Gly Tyr Gly His Cys Ser Ser Glu Ile Ser Asn Ile AspLys Asn Thr 420 425 430 Lys Asn Ala Phe Gln Asp Ile Ser Met Ser Asn ValSer Trp Glu Asn 435 440 445 Ser Gln Thr Glu Tyr Ser Lys Thr Cys Phe IleSer Ser Val Lys His 450 455 460 Thr Gln Ser Glu Asn Gly Asn Lys Asp HisIle Asp Glu Ser Gly Glu 465 470 475 480 Asn Glu Glu Glu Ala Gly Leu GluAsn Ser Ser Glu Ile Ser Ala Asp 485 490 495 Glu Trp Ser Arg Gly Asn IleLeu Lys Asn Ser Val Gly Glu Asn Ile 500 505 510 Glu Pro Val Lys Ile LeuVal Pro Glu Lys Ser Leu Pro Cys Lys Val 515 520 525 Ser Asn Asn Asn TyrPro Ile Pro Glu Gln Met Asn Leu Asn Glu Asp 530 535 540 Ser Cys Asn LysLys Ser Asn Val Ile Asp Asn Lys Ser Gly Lys Val 545 550 555 560 Thr AlaTyr Asp Leu Leu Ser Asn Arg Val Ile Lys Lys Pro Met Ser 565 570 575 AlaSer Ala Leu Phe Val Gln Asp His Arg Pro Gln Phe Leu Ile Glu 580 585 590Asn Pro Lys Thr Ser Leu Glu Asp Ala Thr Leu Gln Ile Glu Glu Leu 595 600605 Trp Lys Thr Leu Ser Glu Glu Glu Lys Leu Lys Tyr Glu Glu Lys Ala 610615 620 Thr Lys Asp Leu Glu Arg Tyr Asn Ser Gln Met Lys Arg Ala Ile Glu625 630 635 640 Gln Glu Ser Gln Met Ser Leu Lys Asp Gly Arg Lys Lys IleLys Pro 645 650 655 Thr Ser Ala Trp Asn Leu Ala Gln Lys His Lys Leu LysThr Ser Leu 660 665 670 Ser Asn Gln Pro Lys Leu Asp Glu Leu Leu Gln SerGln Ile Glu Lys 675 680 685 Arg Arg Ser Gln Asn Ile Lys Met Val Gln IlePro Phe Ser Met Lys 690 695 700 Asn Leu Lys Ile Asn Phe Lys Lys Gln AsnLys Val Asp Leu Glu Glu 705 710 715 720 Lys Asp Glu Pro Cys Leu Ile HisAsn Leu Arg Phe Pro Asp Ala Trp 725 730 735 Leu Met Thr Ser Lys Thr GluVal Met Leu Leu Asn Pro Tyr Arg Val 740 745 750 Glu Glu Ala Leu Leu PheLys Arg Leu Leu Glu Asn His Lys Leu Pro 755 760 765 Ala Glu Pro Leu GluLys Pro Ile Met Leu Thr Glu Ser Leu Phe Asn 770 775 780 Gly Ser His TyrLeu Asp Val Leu Tyr Lys Met Thr Ala Asp Asp Gln 785 790 795 800 Arg TyrSer Gly Ser Thr Tyr Leu Ser Asp Pro Arg Leu Thr Ala Asn 805 810 815 GlyPhe Lys Ile Lys Leu Ile Pro Gly Val Ser Ile Thr Glu Asn Tyr 820 825 830Leu Glu Ile Glu Gly Met Ala Asn Cys Leu Pro Phe Tyr Gly Val Ala 835 840845 Asp Leu Lys Glu Ile Leu Asn Ala Ile Leu Asn Arg Asn Ala Lys Glu 850855 860 Val Tyr Glu Cys Arg Pro Arg Lys Val Ile Ser Tyr Leu Glu Gly Glu865 870 875 880 Ala Val Arg Leu Ser Arg Gln Leu Pro Met Tyr Leu Ser LysGlu Asp 885 890 895 Ile Gln Asp Ile Ile Tyr Arg Met Lys His Gln Phe GlyAsn Glu Ile 900 905 910 Lys Glu Cys Val His Gly Arg Pro Phe Phe His HisLeu Thr Tyr Leu 915 920 925 Pro Glu Thr Thr 930 6 2771 DNA Homo sapiens6 cgaggcggat cgggtgttgc atccatggag cgagctgaga gctcgagtac agaacctgct 60aaggccatca aacctattga tcggaagtca gtccatcaga tttgctctgg gcaggtggta 120ctgagtctaa gcactgcggt aaaggagtta gtagaaaaca gtctggatgc tggtgccact 180aatattgatc taaagcttaa ggactatgga gtggatctta ttgaagtttc agacaatgga 240tgtggggtag aagaagaaaa cttcgaaggc ttaactctga aacatcacac atctaagatt 300caagagtttg ccgacctaac tcaggttgaa acttttggct ttcgggggga agctctgagc 360tcactttgtg cactgagcga tgtcaccatt tctacctgcc acgcatcggc gaaggttgga 420actcgactga tgtttgatca caatgggaaa attatccaga aaacccccta cccccgcccc 480agagggacca cagtcagcgt gcagcagtta ttttccacac tacctgtgcg ccataaggaa 540tttcaaagga atattaagaa ggagtatgcc aaaatggtcc aggtcttaca tgcatactgt 600atcatttcag caggcatccg tgtaagttgc accaatcagc ttggacaagg aaaacgacag 660cctgtggtat gcacaggtgg aagccccagc ataaaggaaa atatcggctc tgtgtttggg 720cagaagcagt tgcaaagcct cattcctttt gttcagctgc cccctagtga ctccgtgtgt 780gaagagtacg gtttgagctg ttcggatgct ctgcataatc ttttttacat ctcaggtttc 840atttcacaat gcacgcatgg agttggaagg agttcaacag acagacagtt tttctttatc 900aaccggcggc cttgtgaccc agcaaaggtc tgcagactcg tgaatgaggt ctaccacatg 960tataatcgac accagtatcc atttgttgtt cttaacattt ctgttgattc agaatgcgtt 1020gatatcaatg ttactccaga taaaaggcaa attttgctac aagaggaaaa gcttttgttg 1080gcagttttaa agacctcttt gataggaatg tttgatagtg atgtcaacaa gctaaatgtc 1140agtcagcagc cactgctgga tgttgaaggt aacttaataa aaatgcatgc agcggatttg 1200gaaaagccca tggtagaaaa gcaggatcaa tccccttcat taaggactgg agaagaaaaa 1260aaagacgtgt ccatttccag actgcgagag gccttttctc ttcgtcacac aacagagaac 1320aagcctcaca gcccaaagac tccagaacca agaaggagcc ctctaggaca gaaaaggggt 1380atgctgtctt ctagcacttc aggtgccatc tctgacaaag gcgtcctgag acctcagaaa 1440gaggcagtga gttccagtca cggacccagt gaccctacgg acagagcgga ggtggagaag 1500gactcggggc acggcagcac ttccgtggat tctgaggggt tcagcatccc agacacgggc 1560agtcactgca gcagcgagta tgcggccagc tccccagggg acaggggctc gcaggaacat 1620gtggactctc aggagaaagc gcctgaaact gacgactctt tttcagatgt ggactgccat 1680tcaaaccagg aagataccgg atgtaaattt cgagttttgc ctcagccaac taatctcgca 1740accccaaaca caaagcgttt taaaaaagaa gaaattcttt ccagttctga catttgtcaa 1800aagttagtaa atactcagga catgtcagcc tctcaggttg atgtagctgt gaaaattaat 1860aagaaagttg tgcccctgga cttttctatg agttctttag ctaaacgaat aaagcagtta 1920catcatgaag cacagcaaag tgaaggggaa cagaattaca ggaagtttag ggcaaagatt 1980tgtcctggag aaaatcaagc agccgaagat gaactaagaa aagagataag taaaacgatg 2040tttgcagaaa tggaaatcat tggtcagttt aacctgggat ttataataac caaactgaat 2100gaggatatct tcatagtgga ccagcatgcc acggacgaga agtataactt cgagatgctg 2160cagcagcaca ccgtgctcca ggggcagagg ctcatagcac ctcagactct caacttaact 2220gctgttaatg aagctgttct gatagaaaat ctggaaatat ttagaaagaa tggctttgat 2280tttgttatcg atgaaaatgc tccagtcact gaaagggcta aactgatttc cttgccaact 2340agtaaaaact ggaccttcgg accccaggac gtcgatgaac tgatcttcat gctgagcgac 2400agccctgggg tcatgtgccg gccttcccga gtcaagcaga tgtttgcctc cagagcctgc 2460cggaagtcgg tgatgattgg gactgctctt aacacaagcg agatgaagaa actgatcacc 2520cacatggggg agatggacca cccctggaac tgtccccatg gaaggccaac catgagacac 2580atcgccaacc tgggtgtcat ttctcagaac tgaccgtagt cactgtatgg aataattggt 2640tttatcgcag atttttatgt tttgaaagac agagtcttca ctaacctttt ttgttttaaa 2700atgaaacctg ctacttaaaa aaaatacaca tcacacccat ttaaaagtga tcttgagaac 2760cttttcaaac c 2771 7 932 PRT Homo sapiens 7 Met Lys Gln Leu Pro Ala AlaThr Val Arg Leu Leu Ser Ser Ser Gln 1 5 10 15 Ile Ile Thr Ser Val ValSer Val Val Lys Glu Leu Ile Glu Asn Ser 20 25 30 Leu Asp Ala Gly Ala ThrSer Val Asp Val Lys Leu Glu Asn Tyr Gly 35 40 45 Phe Asp Lys Ile Glu ValArg Asp Asn Gly Glu Gly Ile Lys Ala Val 50 55 60 Asp Ala Pro Val Met AlaMet Lys Tyr Tyr Thr Ser Lys Ile Asn Ser 65 70 75 80 His Glu Asp Leu GluAsn Leu Thr Thr Tyr Gly Phe Arg Gly Glu Ala 85 90 95 Leu Gly Ser Ile CysCys Ile Ala Glu Val Leu Ile Thr Thr Arg Thr 100 105 110 Ala Ala Asp AsnPhe Ser Thr Gln Tyr Val Leu Asp Gly Ser Gly His 115 120 125 Ile Leu SerGln Lys Pro Ser His Leu Gly Gln Gly Thr Thr Val Thr 130 135 140 Ala LeuArg Leu Phe Lys Asn Leu Pro Val Arg Lys Gln Phe Tyr Ser 145 150 155 160Thr Ala Lys Lys Cys Lys Asp Glu Ile Lys Lys Ile Gln Asp Leu Leu 165 170175 Met Ser Phe Gly Ile Leu Lys Pro Asp Leu Arg Ile Val Phe Val His 180185 190 Asn Lys Ala Val Ile Trp Gln Lys Ser Arg Val Ser Asp His Lys Met195 200 205 Ala Leu Met Ser Val Leu Gly Thr Ala Val Met Asn Asn Met GluSer 210 215 220 Phe Gln Tyr His Ser Glu Glu Ser Gln Ile Tyr Leu Ser GlyPhe Leu 225 230 235 240 Pro Lys Cys Asp Ala Asp His Ser Phe Thr Ser LeuSer Thr Pro Glu 245 250 255 Arg Ser Phe Ile Phe Ile Asn Ser Arg Pro ValHis Gln Lys Asp Ile 260 265 270 Leu Lys Leu Ile Arg His His Tyr Asn LeuLys Cys Leu Lys Glu Ser 275 280 285 Thr Arg Leu Tyr Pro Val Phe Phe LeuLys Ile Asp Val Pro Thr Ala 290 295 300 Asp Val Asp Val Asn Leu Thr ProAsp Lys Ser Gln Val Leu Leu Gln 305 310 315 320 Asn Lys Glu Ser Val LeuIle Ala Leu Glu Asn Leu Met Thr Thr Cys 325 330 335 Tyr Gly Pro Leu ProSer Thr Asn Ser Tyr Glu Asn Asn Lys Thr Asp 340 345 350 Val Ser Ala AlaAsp Ile Val Leu Ser Lys Thr Ala Glu Thr Asp Val 355 360 365 Leu Phe AsnLys Val Glu Ser Ser Gly Lys Asn Tyr Ser Asn Val Asp 370 375 380 Thr SerVal Ile Pro Phe Gln Asn Asp Met His Asn Asp Glu Ser Gly 385 390 395 400Lys Asn Thr Asp Asp Cys Leu Asn His Gln Ile Ser Ile Gly Asp Phe 405 410415 Gly Tyr Gly His Cys Ser Ser Glu Ile Ser Asn Ile Asp Lys Asn Thr 420425 430 Lys Asn Ala Phe Gln Asp Ile Ser Met Ser Asn Val Ser Trp Glu Asn435 440 445 Ser Gln Thr Glu Tyr Ser Lys Thr Cys Phe Ile Ser Ser Val LysHis 450 455 460 Thr Gln Ser Glu Asn Gly Asn Lys Asp His Ile Asp Glu SerGly Glu 465 470 475 480 Asn Glu Glu Glu Ala Gly Leu Glu Asn Ser Ser GluIle Ser Ala Asp 485 490 495 Glu Trp Ser Arg Gly Asn Ile Leu Lys Asn SerVal Gly Glu Asn Ile 500 505 510 Glu Pro Val Lys Ile Leu Val Pro Glu LysSer Leu Pro Cys Lys Val 515 520 525 Ser Asn Asn Asn Tyr Pro Ile Pro GluGln Met Asn Leu Asn Glu Asp 530 535 540 Ser Cys Asn Lys Lys Ser Asn ValIle Asp Asn Lys Ser Gly Lys Val 545 550 555 560 Thr Ala Tyr Asp Leu LeuSer Asn Arg Val Ile Lys Lys Pro Met Ser 565 570 575 Ala Ser Ala Leu PheVal Gln Asp His Arg Pro Gln Phe Leu Ile Glu 580 585 590 Asn Pro Lys ThrSer Leu Glu Asp Ala Thr Leu Gln Ile Glu Glu Leu 595 600 605 Trp Lys ThrLeu Ser Glu Glu Glu Lys Leu Lys Tyr Glu Glu Lys Ala 610 615 620 Thr LysAsp Leu Glu Arg Tyr Asn Ser Gln Met Lys Arg Ala Ile Glu 625 630 635 640Gln Glu Ser Gln Met Ser Leu Lys Asp Gly Arg Lys Lys Ile Lys Pro 645 650655 Thr Ser Ala Trp Asn Leu Ala Gln Lys His Lys Leu Lys Thr Ser Leu 660665 670 Ser Asn Gln Pro Lys Leu Asp Glu Leu Leu Gln Ser Gln Ile Glu Lys675 680 685 Arg Arg Ser Gln Asn Ile Lys Met Val Gln Ile Pro Phe Ser MetLys 690 695 700 Asn Leu Lys Ile Asn Phe Lys Lys Gln Asn Lys Val Asp LeuGlu Glu 705 710 715 720 Lys Asp Glu Pro Cys Leu Ile His Asn Leu Arg PhePro Asp Ala Trp 725 730 735 Leu Met Thr Ser Lys Thr Glu Val Met Leu LeuAsn Pro Tyr Arg Val 740 745 750 Glu Glu Ala Leu Leu Phe Lys Arg Leu LeuGlu Asn His Lys Leu Pro 755 760 765 Ala Glu Pro Leu Glu Lys Pro Ile MetLeu Thr Glu Ser Leu Phe Asn 770 775 780 Gly Ser His Tyr Leu Asp Val LeuTyr Lys Met Thr Ala Asp Asp Gln 785 790 795 800 Arg Tyr Ser Gly Ser ThrTyr Leu Ser Asp Pro Arg Leu Thr Ala Asn 805 810 815 Gly Phe Lys Ile LysLeu Ile Pro Gly Val Ser Ile Thr Glu Asn Tyr 820 825 830 Leu Glu Ile GluGly Met Ala Asn Cys Leu Pro Phe Tyr Gly Val Ala 835 840 845 Asp Leu LysGlu Ile Leu Asn Ala Ile Leu Asn Arg Asn Ala Lys Glu 850 855 860 Val TyrGlu Cys Arg Pro Arg Lys Val Ile Ser Tyr Leu Glu Gly Glu 865 870 875 880Ala Val Arg Leu Ser Arg Gln Leu Pro Met Tyr Leu Ser Lys Glu Asp 885 890895 Ile Gln Asp Ile Ile Tyr Arg Met Lys His Gln Phe Gly Asn Glu Ile 900905 910 Lys Glu Cys Val His Gly Arg Pro Phe Phe His His Leu Thr Tyr Leu915 920 925 Pro Glu Thr Thr 930 8 3063 DNA Homo sapiens 8 ggcacgagtggctgcttgcg gctagtggat ggtaattgcc tgcctcgcgc tagcagcaag 60 ctgctctgttaaaagcgaaa atgaaacaat tgcctgcggc aacagttcga ctcctttcaa 120 gttctcagatcatcacttcg gtggtcagtg ttgtaaaaga gcttattgaa aactccttgg 180 atgctggtgccacaagcgta gatgttaaac tggagaacta tggatttgat aaaattgagg 240 tgcgagataacggggagggt atcaaggctg ttgatgcacc tgtaatggca atgaagtact 300 acacctcaaaaataaatagt catgaagatc ttgaaaattt gacaacttac ggttttcgtg 360 gagaagccttggggtcaatt tgttgtatag ctgaggtttt aattacaaca agaacggctg 420 ctgataattttagcacccag tatgttttag atggcagtgg ccacatactt tctcagaaac 480 cttcacatcttggtcaaggt acaactgtaa ctgctttaag attatttaag aatctacctg 540 taagaaagcagttttactca actgcaaaaa aatgtaaaga tgaaataaaa aagatccaag 600 atctcctcatgagctttggt atccttaaac ctgacttaag gattgtcttt gtacataaca 660 aggcagttatttggcagaaa agcagagtat cagatcacaa gatggctctc atgtcagttc 720 tggggactgctgttatgaac aatatggaat cctttcagta ccactctgaa gaatctcaga 780 tttatctcagtggatttctt ccaaagtgtg atgcagacca ctctttcact agtctttcaa 840 caccagaaagaagtttcatc ttcataaaca gtcgaccagt acatcaaaaa gatatcttaa 900 agttaatccgacatcattac aatctgaaat gcctaaagga atctactcgt ttgtatcctg 960 ttttctttctgaaaatcgat gttcctacag ctgatgttga tgtaaattta acaccagata 1020 aaagccaagtattattacaa aataaggaat ctgttttaat tgctcttgaa aatctgatga 1080 cgacttgttatggaccatta cctagtacaa attcttatga aaataataaa acagatgttt 1140 ccgcagctgacatcgttctt agtaaaacag cagaaacaga tgtgcttttt aataaagtgg 1200 aatcatctggaaagaattat tcaaatgttg atacttcagt cattccattc caaaatgata 1260 tgcataatgatgaatctgga aaaaacactg atgattgttt aaatcaccag ataagtattg 1320 gtgactttggttatggtcat tgtagtagtg aaatttctaa cattgataaa aacactaaga 1380 atgcatttcaggacatttca atgagtaatg tatcatggga gaactctcag acggaatata 1440 gtaaaacttgttttataagt tccgttaagc acacccagtc agaaaatggc aataaagacc 1500 atatagatgagagtggggaa aatgaggaag aagcaggtct tgaaaactct tcggaaattt 1560 ctgcagatgagtggagcagg ggaaatatac ttaaaaattc agtgggagag aatattgaac 1620 ctgtgaaaattttagtgcct gaaaaaagtt taccatgtaa agtaagtaat aataattatc 1680 caatccctgaacaaatgaat cttaatgaag attcatgtaa caaaaaatca aatgtaatag 1740 ataataaatctggaaaagtt acagcttatg atttacttag caatcgagta atcaagaaac 1800 ccatgtcagcaagtgctctt tttgttcaag atcatcgtcc tcagtttctc atagaaaatc 1860 ctaagactagtttagaggat gcaacactac aaattgaaga actgtggaag acattgagtg 1920 aagaggaaaaactgaaatat gaagagaagg ctactaaaga cttggaacga tacaatagtc 1980 aaatgaagagagccattgaa caggagtcac aaatgtcact aaaagatggc agaaaaaaga 2040 taaaacccaccagcgcatgg aatttggccc agaagcacaa gttaaaaacc tcattatcta 2100 atcaaccaaaacttgatgaa ctccttcagt cccaaattga aaaaagaagg agtcaaaata 2160 ttaaaatggtacagatcccc ttttctatga aaaacttaaa aataaatttt aagaaacaaa 2220 acaaagttgacttagaagag aaggatgaac cttgcttgat ccacaatctc aggtttcctg 2280 atgcatggctaatgacatcc aaaacagagg taatgttatt aaatccatat agagtagaag 2340 aagccctgctatttaaaaga cttcttgaga atcataaact tcctgcagag ccactggaaa 2400 agccaattatgttaacagag agtcttttta atggatctca ttatttagac gttttatata 2460 aaatgacagcagatgaccaa agatacagtg gatcaactta cctgtctgat cctcgtctta 2520 cagcgaatggtttcaagata aaattgatac caggagtttc aattactgaa aattacttgg 2580 aaatagaaggaatggctaat tgtctcccat tctatggagt agcagattta aaagaaattc 2640 ttaatgctatattaaacaga aatgcaaagg aagtttatga atgtagacct cgcaaagtga 2700 taagttatttagagggagaa gcagtgcgtc tatccagaca attacccatg tacttatcaa 2760 aagaggacatccaagacatt atctacagaa tgaagcacca gtttggaaat gaaattaaag 2820 agtgtgttcatggtcgccca ttttttcatc atttaaccta tcttccagaa actacatgat 2880 taaatatgtttaagaagatt agttaccatt gaaattggtt ctgtcataaa acagcatgag 2940 tctggttttaaattatcttt gtattatgtg tcacatggtt attttttaaa tgaggattca 3000 ctgacttgtttttatattga aaaaagttcc acgtattgta gaaaacgtaa ataaactaat 3060 aac 3063 9934 PRT Homo sapiens 9 Met Ala Val Gln Pro Lys Glu Thr Leu Gln Leu GluSer Ala Ala Glu 1 5 10 15 Val Gly Phe Val Arg Phe Phe Gln Gly Met ProGlu Lys Pro Thr Thr 20 25 30 Thr Val Arg Leu Phe Asp Arg Gly Asp Phe TyrThr Ala His Gly Glu 35 40 45 Asp Ala Leu Leu Ala Ala Arg Glu Val Phe LysThr Gln Gly Val Ile 50 55 60 Lys Tyr Met Gly Pro Ala Gly Ala Lys Asn LeuGln Ser Val Val Leu 65 70 75 80 Ser Lys Met Asn Phe Glu Ser Phe Val LysAsp Leu Leu Leu Val Arg 85 90 95 Gln Tyr Arg Val Glu Val Tyr Lys Asn ArgAla Gly Asn Lys Ala Ser 100 105 110 Lys Glu Asn Asp Trp Tyr Leu Ala TyrLys Ala Ser Pro Gly Asn Leu 115 120 125 Ser Gln Phe Glu Asp Ile Leu PheGly Asn Asn Asp Met Ser Ala Ser 130 135 140 Ile Gly Val Val Gly Val LysMet Ser Ala Val Asp Gly Gln Arg Gln 145 150 155 160 Val Gly Val Gly TyrVal Asp Ser Ile Gln Arg Lys Leu Gly Leu Cys 165 170 175 Glu Phe Pro AspAsn Asp Gln Phe Ser Asn Leu Glu Ala Leu Leu Ile 180 185 190 Gln Ile GlyPro Lys Glu Cys Val Leu Pro Gly Gly Glu Thr Ala Gly 195 200 205 Asp MetGly Lys Leu Arg Gln Ile Ile Gln Arg Gly Gly Ile Leu Ile 210 215 220 ThrGlu Arg Lys Lys Ala Asp Phe Ser Thr Lys Asp Ile Tyr Gln Asp 225 230 235240 Leu Asn Arg Leu Leu Lys Gly Lys Lys Gly Glu Gln Met Asn Ser Ala 245250 255 Val Leu Pro Glu Met Glu Asn Gln Val Ala Val Ser Ser Leu Ser Ala260 265 270 Val Ile Lys Phe Leu Glu Leu Leu Ser Asp Asp Ser Asn Phe GlyGln 275 280 285 Phe Glu Leu Thr Thr Phe Asp Phe Ser Gln Tyr Met Lys LeuAsp Ile 290 295 300 Ala Ala Val Arg Ala Leu Asn Leu Phe Gln Gly Ser ValGlu Asp Thr 305 310 315 320 Thr Gly Ser Gln Ser Leu Ala Ala Leu Leu AsnLys Cys Lys Thr Pro 325 330 335 Gln Gly Gln Arg Leu Val Asn Gln Trp IleLys Gln Pro Leu Met Asp 340 345 350 Lys Asn Arg Ile Glu Glu Arg Leu AsnLeu Val Glu Ala Phe Val Glu 355 360 365 Asp Ala Glu Leu Arg Gln Thr LeuGln Glu Asp Leu Leu Arg Arg Phe 370 375 380 Pro Asp Leu Asn Arg Leu AlaLys Lys Phe Gln Arg Gln Ala Ala Asn 385 390 395 400 Leu Gln Asp Cys TyrArg Leu Tyr Gln Gly Ile Asn Gln Leu Pro Asn 405 410 415 Val Ile Gln AlaLeu Glu Lys His Glu Gly Lys His Gln Lys Leu Leu 420 425 430 Leu Ala ValPhe Val Thr Pro Leu Thr Asp Leu Arg Ser Asp Phe Ser 435 440 445 Lys PheGln Glu Met Ile Glu Thr Thr Leu Asp Met Asp Gln Val Glu 450 455 460 AsnHis Glu Phe Leu Val Lys Pro Ser Phe Asp Pro Asn Leu Ser Glu 465 470 475480 Leu Arg Glu Ile Met Asn Asp Leu Glu Lys Lys Met Gln Ser Thr Leu 485490 495 Ile Ser Ala Ala Arg Asp Leu Gly Leu Asp Pro Gly Lys Gln Ile Lys500 505 510 Leu Asp Ser Ser Ala Gln Phe Gly Tyr Tyr Phe Arg Val Thr CysLys 515 520 525 Glu Glu Lys Val Leu Arg Asn Asn Lys Asn Phe Ser Thr ValAsp Ile 530 535 540 Gln Lys Asn Gly Val Lys Phe Thr Asn Ser Lys Leu ThrSer Leu Asn 545 550 555 560 Glu Glu Tyr Thr Lys Asn Lys Thr Glu Tyr GluGlu Ala Gln Asp Ala 565 570 575 Ile Val Lys Glu Ile Val Asn Ile Ser SerGly Tyr Val Glu Pro Met 580 585 590 Gln Thr Leu Asn Asp Val Leu Ala GlnLeu Asp Ala Val Val Ser Phe 595 600 605 Ala His Val Ser Asn Gly Ala ProVal Pro Tyr Val Arg Pro Ala Ile 610 615 620 Leu Glu Lys Gly Gln Gly ArgIle Ile Leu Lys Ala Ser Arg His Ala 625 630 635 640 Cys Val Glu Val GlnAsp Glu Ile Ala Phe Ile Pro Asn Asp Val Tyr 645 650 655 Phe Glu Lys AspLys Gln Met Phe His Ile Ile Thr Gly Pro Asn Met 660 665 670 Gly Gly LysSer Thr Tyr Ile Arg Gln Thr Gly Val Ile Val Leu Met 675 680 685 Ala GlnIle Gly Cys Phe Val Pro Cys Glu Ser Ala Glu Val Ser Ile 690 695 700 ValAsp Cys Ile Leu Ala Arg Val Gly Ala Gly Asp Ser Gln Leu Lys 705 710 715720 Gly Val Ser Thr Phe Met Ala Glu Met Leu Glu Thr Ala Ser Ile Leu 725730 735 Arg Ser Ala Thr Lys Asp Ser Leu Ile Ile Ile Asp Glu Leu Gly Arg740 745 750 Gly Thr Ser Thr Tyr Asp Gly Phe Gly Leu Ala Trp Ala Ile SerGlu 755 760 765 Tyr Ile Ala Thr Lys Ile Gly Ala Phe Cys Met Phe Ala ThrHis Phe 770 775 780 His Glu Leu Thr Ala Leu Ala Asn Gln Ile Pro Thr ValAsn Asn Leu 785 790 795 800 His Val Thr Ala Leu Thr Thr Glu Glu Thr LeuThr Met Leu Tyr Gln 805 810 815 Val Lys Lys Gly Val Cys Asp Gln Ser PheGly Ile His Val Ala Glu 820 825 830 Leu Ala Asn Phe Pro Lys His Val IleGlu Cys Ala Lys Gln Lys Ala 835 840 845 Leu Glu Leu Glu Glu Phe Gln TyrIle Gly Glu Ser Gln Gly Tyr Asp 850 855 860 Ile Met Glu Pro Ala Ala LysLys Cys Tyr Leu Glu Arg Glu Gln Gly 865 870 875 880 Glu Lys Ile Ile GlnGlu Phe Leu Ser Lys Val Lys Gln Met Pro Phe 885 890 895 Thr Glu Met SerGlu Glu Asn Ile Thr Ile Lys Leu Lys Gln Leu Lys 900 905 910 Ala Glu ValIle Ala Lys Asn Asn Ser Phe Val Asn Glu Ile Ile Ser 915 920 925 Arg IleLys Val Thr Thr 930 10 3145 DNA Homo sapiens 10 ggcgggaaac agcttagtgggtgtggggtc gcgcattttc ttcaaccagg aggtgaggag 60 gtttcgacat ggcggtgcagccgaaggaga cgctgcagtt ggagagcgcg gccgaggtcg 120 gcttcgtgcg cttctttcagggcatgccgg agaagccgac caccacagtg cgccttttcg 180 accggggcga cttctatacggcgcacggcg aggacgcgct gctggccgcc cgggaggtgt 240 tcaagaccca gggggtgatcaagtacatgg ggccggcagg agcaaagaat ctgcagagtg 300 ttgtgcttag taaaatgaattttgaatctt ttgtaaaaga tcttcttctg gttcgtcagt 360 atagagttga agtttataagaatagagctg gaaataaggc atccaaggag aatgattggt 420 atttggcata taaggcttctcctggcaatc tctctcagtt tgaagacatt ctctttggta 480 acaatgatat gtcagcttccattggtgttg tgggtgttaa aatgtccgca gttgatggcc 540 agagacaggt tggagttgggtatgtggatt ccatacagag gaaactagga ctgtgtgaat 600 tccctgataa tgatcagttctccaatcttg aggctctcct catccagatt ggaccaaagg 660 aatgtgtttt acccggaggagagactgctg gagacatggg gaaactgaga cagataattc 720 aaagaggagg aattctgatcacagaaagaa aaaaagctga cttttccaca aaagacattt 780 atcaggacct caaccggttgttgaaaggca aaaagggaga gcagatgaat agtgctgtat 840 tgccagaaat ggagaatcaggttgcagttt catcactgtc tgcggtaatc aagtttttag 900 aactcttatc agatgattccaactttggac agtttgaact gactactttt gacttcagcc 960 agtatatgaa attggatattgcagcagtca gagcccttaa cctttttcag ggttctgttg 1020 aagataccac tggctctcagtctctggctg ccttgctgaa taagtgtaaa acccctcaag 1080 gacaaagact tgttaaccagtggattaagc agcctctcat ggataagaac agaatagagg 1140 agagattgaa tttagtggaagcttttgtag aagatgcaga attgaggcag actttacaag 1200 aagatttact tcgtcgattcccagatctta accgacttgc caagaagttt caaagacaag 1260 cagcaaactt acaagattgttaccgactct atcagggtat aaatcaacta cctaatgtta 1320 tacaggctct ggaaaaacatgaaggaaaac accagaaatt attgttggca gtttttgtga 1380 ctcctcttac tgatcttcgttctgacttct ccaagtttca ggaaatgata gaaacaactt 1440 tagatatgga tcaggtggaaaaccatgaat tccttgtaaa accttcattt gatcctaatc 1500 tcagtgaatt aagagaaataatgaatgact tggaaaagaa gatgcagtca acattaataa 1560 gtgcagccag agatcttggcttggaccctg gcaaacagat taaactggat tccagtgcac 1620 agtttggata ttactttcgtgtaacctgta aggaagaaaa agtccttcgt aacaataaaa 1680 actttagtac tgtagatatccagaagaatg gtgttaaatt taccaacagc aaattgactt 1740 ctttaaatga agagtataccaaaaataaaa cagaatatga agaagcccag gatgccattg 1800 ttaaagaaat tgtcaatatttcttcaggct atgtagaacc aatgcagaca ctcaatgatg 1860 tgttagctca gctagatgctgttgtcagct ttgctcacgt gtcaaatgga gcacctgttc 1920 catatgtacg accagccattttggagaaag gacaaggaag aattatatta aaagcatcca 1980 ggcatgcttg tgttgaagttcaagatgaaa ttgcatttat tcctaatgac gtatactttg 2040 aaaaagataa acagatgttccacatcatta ctggccccaa tatgggaggt aaatcaacat 2100 atattcgaca aactggggtgatagtactca tggcccaaat tgggtgtttt gtgccatgtg 2160 agtcagcaga agtgtccattgtggactgca tcttagcccg agtaggggct ggtgacagtc 2220 aattgaaagg agtctccacgttcatggctg aaatgttgga aactgcttct atcctcaggt 2280 ctgcaaccaa agattcattaataatcatag atgaattggg aagaggaact tctacctacg 2340 atggatttgg gttagcatgggctatatcag aatacattgc aacaaagatt ggtgcttttt 2400 gcatgtttgc aacccattttcatgaactta ctgccttggc caatcagata ccaactgtta 2460 ataatctaca tgtcacagcactcaccactg aagagacctt aactatgctt tatcaggtga 2520 agaaaggtgt ctgtgatcaaagttttggga ttcatgttgc agagcttgct aatttcccta 2580 agcatgtaat agagtgtgctaaacagaaag ccctggaact tgaggagttt cagtatattg 2640 gagaatcgca aggatatgatatcatggaac cagcagcaaa gaagtgctat ctggaaagag 2700 agcaaggtga aaaaattattcaggagttcc tgtccaaggt gaaacaaatg ccctttactg 2760 aaatgtcaga agaaaacatcacaataaagt taaaacagct aaaagctgaa gtaatagcaa 2820 agaataatag ctttgtaaatgaaatcattt cacgaataaa agttactacg tgaaaaatcc 2880 cagtaatgga atgaaggtaatattgataag ctattgtctg taatagtttt atattgtttt 2940 atattaaccc tttttccatagtgttaactg tcagtgccca tgggctatca acttaataag 3000 atatttagta atattttactttgaggacat tttcaaagat ttttattttg aaaaatgaga 3060 gctgtaactg aggactgtttgcaattgaca taggcaataa taagtgatgt gctgaatttt 3120 ataaataaaa tcatgtagtttgtgg 3145 11 756 PRT Homo sapiens 11 Met Ser Phe Val Ala Gly Val IleArg Arg Leu Asp Glu Thr Val Val 1 5 10 15 Asn Arg Ile Ala Ala Gly GluVal Ile Gln Arg Pro Ala Asn Ala Ile 20 25 30 Lys Glu Met Ile Glu Asn CysLeu Asp Ala Lys Ser Thr Ser Ile Gln 35 40 45 Val Ile Val Lys Glu Gly GlyLeu Lys Leu Ile Gln Ile Gln Asp Asn 50 55 60 Gly Thr Gly Ile Arg Lys GluAsp Leu Asp Ile Val Cys Glu Arg Phe 65 70 75 80 Thr Thr Ser Lys Leu GlnSer Phe Glu Asp Leu Ala Ser Ile Ser Thr 85 90 95 Tyr Gly Phe Arg Gly GluAla Leu Ala Ser Ile Ser His Val Ala His 100 105 110 Val Thr Ile Thr ThrLys Thr Ala Asp Gly Lys Cys Ala Tyr Arg Ala 115 120 125 Ser Tyr Ser AspGly Lys Leu Lys Ala Pro Pro Lys Pro Cys Ala Gly 130 135 140 Asn Gln GlyThr Gln Ile Thr Val Glu Asp Leu Phe Tyr Asn Ile Ala 145 150 155 160 ThrArg Arg Lys Ala Leu Lys Asn Pro Ser Glu Glu Tyr Gly Lys Ile 165 170 175Leu Glu Val Val Gly Arg Tyr Ser Val His Asn Ala Gly Ile Ser Phe 180 185190 Ser Val Lys Lys Gln Gly Glu Thr Val Ala Asp Val Arg Thr Leu Pro 195200 205 Asn Ala Ser Thr Val Asp Asn Ile Arg Ser Ile Phe Gly Asn Ala Val210 215 220 Ser Arg Glu Leu Ile Glu Ile Gly Cys Glu Asp Lys Thr Leu AlaPhe 225 230 235 240 Lys Met Asn Gly Tyr Ile Ser Asn Ala Asn Tyr Ser ValLys Lys Cys 245 250 255 Ile Phe Leu Leu Phe Ile Asn His Arg Leu Val GluSer Thr Ser Leu 260 265 270 Arg Lys Ala Ile Glu Thr Val Tyr Ala Ala TyrLeu Pro Lys Asn Thr 275 280 285 His Pro Phe Leu Tyr Leu Ser Leu Glu IleSer Pro Gln Asn Val Asp 290 295 300 Val Asn Val His Pro Thr Lys His GluVal His Phe Leu His Glu Glu 305 310 315 320 Ser Ile Leu Glu Arg Val GlnGln His Ile Glu Ser Lys Leu Leu Gly 325 330 335 Ser Asn Ser Ser Arg MetTyr Phe Thr Gln Thr Leu Leu Pro Gly Leu 340 345 350 Ala Gly Pro Ser GlyGlu Met Val Lys Ser Thr Thr Ser Leu Thr Ser 355 360 365 Ser Ser Thr SerGly Ser Ser Asp Lys Val Tyr Ala His Gln Met Val 370 375 380 Arg Thr AspSer Arg Glu Gln Lys Leu Asp Ala Phe Leu Gln Pro Leu 385 390 395 400 SerLys Pro Leu Ser Ser Gln Pro Gln Ala Ile Val Thr Glu Asp Lys 405 410 415Thr Asp Ile Ser Ser Gly Arg Ala Arg Gln Gln Asp Glu Glu Met Leu 420 425430 Glu Leu Pro Ala Pro Ala Glu Val Ala Ala Lys Asn Gln Ser Leu Glu 435440 445 Gly Asp Thr Thr Lys Gly Thr Ser Glu Met Ser Glu Lys Arg Gly Pro450 455 460 Thr Ser Ser Asn Pro Arg Lys Arg His Arg Glu Asp Ser Asp ValGlu 465 470 475 480 Met Val Glu Asp Asp Ser Arg Lys Glu Met Thr Ala AlaCys Thr Pro 485 490 495 Arg Arg Arg Ile Ile Asn Leu Thr Ser Val Leu SerLeu Gln Glu Glu 500 505 510 Ile Asn Glu Gln Gly His Glu Val Leu Arg GluMet Leu His Asn His 515 520 525 Ser Phe Val Gly Cys Val Asn Pro Gln TrpAla Leu Ala Gln His Gln 530 535 540 Thr Lys Leu Tyr Leu Leu Asn Thr ThrLys Leu Ser Glu Glu Leu Phe 545 550 555 560 Tyr Gln Ile Leu Ile Tyr AspPhe Ala Asn Phe Gly Val Leu Arg Leu 565 570 575 Ser Glu Pro Ala Pro LeuPhe Asp Leu Ala Met Leu Ala Leu Asp Ser 580 585 590 Pro Glu Ser Gly TrpThr Glu Glu Asp Gly Pro Lys Glu Gly Leu Ala 595 600 605 Glu Tyr Ile ValGlu Phe Leu Lys Lys Lys Ala Glu Met Leu Ala Asp 610 615 620 Tyr Phe SerLeu Glu Ile Asp Glu Glu Gly Asn Leu Ile Gly Leu Pro 625 630 635 640 LeuLeu Ile Asp Asn Tyr Val Pro Pro Leu Glu Gly Leu Pro Ile Phe 645 650 655Ile Leu Arg Leu Ala Thr Glu Val Asn Trp Asp Glu Glu Lys Glu Cys 660 665670 Phe Glu Ser Leu Ser Lys Glu Cys Ala Met Phe Tyr Ser Ile Arg Lys 675680 685 Gln Tyr Ile Ser Glu Glu Ser Thr Leu Ser Gly Gln Gln Ser Glu Val690 695 700 Pro Gly Ser Ile Pro Asn Ser Trp Lys Trp Thr Val Glu His IleVal 705 710 715 720 Tyr Lys Ala Leu Arg Ser His Ile Leu Pro Pro Lys HisPhe Thr Glu 725 730 735 Asp Gly Asn Ile Leu Gln Leu Ala Asn Leu Pro AspLeu Tyr Lys Val 740 745 750 Phe Glu Arg Cys 755 12 2484 DNA Homo sapiens12 cttggctctt ctggcgccaa aatgtcgttc gtggcagggg ttattcggcg gctggacgag 60acagtggtga accgcatcgc ggcgggggaa gttatccagc ggccagctaa tgctatcaaa 120gagatgattg agaactgttt agatgcaaaa tccacaagta ttcaagtgat tgttaaagag 180ggaggcctga agttgattca gatccaagac aatggcaccg ggatcaggaa agaagatctg 240gatattgtat gtgaaaggtt cactactagt aaactgcagt cctttgagga tttagccagt 300atttctacct atggctttcg aggtgaggct ttggccagca taagccatgt ggctcatgtt 360actattacaa cgaaaacagc tgatggaaag tgtgcataca gagcaagtta ctcagatgga 420aaactgaaag cccctcctaa accatgtgct ggcaatcaag ggacccagat cacggtggag 480gacctttttt acaacatagc cacgaggaga aaagctttaa aaaatccaag tgaagaatat 540gggaaaattt tggaagttgt tggcaggtat tcagtacaca atgcaggcat tagtttctca 600gttaaaaaac aaggagagac agtagctgat gttaggacac tacccaatgc ctcaaccgtg 660gacaatattc gctccatctt tggaaatgct gttagtcgag aactgataga aattggatgt 720gaggataaaa ccctagcctt caaaatgaat ggttacatat ccaatgcaaa ctactcagtg 780aagaagtgca tcttcttact cttcatcaac catcgtctgg tagaatcaac ttccttgaga 840aaagccatag aaacagtgta tgcagcctat ttgcccaaaa acacacaccc attcctgtac 900ctcagtttag aaatcagtcc ccagaatgtg gatgttaatg tgcaccccac aaagcatgaa 960gttcacttcc tgcacgagga gagcatcctg gagcgggtgc agcagcacat cgagagcaag 1020ctcctgggct ccaattcctc caggatgtac ttcacccaga ctttgctacc aggacttgct 1080ggcccctctg gggagatggt taaatccaca acaagtctga cctcgtcttc tacttctgga 1140agtagtgata aggtctatgc ccaccagatg gttcgtacag attcccggga acagaagctt 1200gatgcatttc tgcagcctct gagcaaaccc ctgtccagtc agccccaggc cattgtcaca 1260gaggataaga cagatatttc tagtggcagg gctaggcagc aagatgagga gatgcttgaa 1320ctcccagccc ctgctgaagt ggctgccaaa aatcagagct tggaggggga tacaacaaag 1380gggacttcag aaatgtcaga gaagagagga cctacttcca gcaaccccag aaagagacat 1440cgggaagatt ctgatgtgga aatggtggaa gatgattccc gaaaggaaat gactgcagct 1500tgtacccccc ggagaaggat cattaacctc actagtgttt tgagtctcca ggaagaaatt 1560aatgagcagg gacatgaggt tctccgggag atgttgcata accactcctt cgtgggctgt 1620gtgaatcctc agtgggcctt ggcacagcat caaaccaagt tataccttct caacaccacc 1680aagcttagtg aagaactgtt ctaccagata ctcatttatg attttgccaa ttttggtgtt 1740ctcaggttat cggagccagc accgctcttt gaccttgcca tgcttgcctt agatagtcca 1800gagagtggct ggacagagga agatggtccc aaagaaggac ttgctgaata cattgttgag 1860tttctgaaga agaaggctga gatgcttgca gactatttct ctttggaaat tgatgaggaa 1920gggaacctga ttggattacc ccttctgatt gacaactatg tgcccccttt ggagggactg 1980cctatcttca ttcttcgact agccactgag gtgaattggg acgaagaaaa ggaatgtttt 2040gaaagcctca gtaaagaatg cgctatgttc tattccatcc ggaagcagta catatctgag 2100gagtcgaccc tctcaggcca gcagagtgaa gtgcctggct ccattccaaa ctcctggaag 2160tggactgtgg aacacattgt ctataaagcc ttgcgctcac acattctgcc tcctaaacat 2220ttcacagaag atggaaatat cctgcagctt gctaacctgc ctgatctata caaagtcttt 2280gagaggtgtt aaatatggtt atttatgcac tgtgggatgt gttcttcttt ctctgtattc 2340cgatacaaag tgttgtatca aagtgtgata tacaaagtgt accaacataa gtgttggtag 2400cacttaagac ttatacttgc cttctgatag tattccttta tacacagtgg attgattata 2460aataaataga tgtgtcttaa cata 2484 13 133 PRT Homo sapiens 13 Met Lys GlnLeu Pro Ala Ala Thr Val Arg Leu Leu Ser Ser Ser Gln 1 5 10 15 Ile IleThr Ser Val Val Ser Val Val Lys Glu Leu Ile Glu Asn Ser 20 25 30 Leu AspAla Gly Ala Thr Ser Val Asp Val Lys Leu Glu Asn Tyr Gly 35 40 45 Phe AspLys Ile Glu Val Arg Asp Asn Gly Glu Gly Ile Lys Ala Val 50 55 60 Asp AlaPro Val Met Ala Met Lys Tyr Tyr Thr Ser Lys Ile Asn Ser 65 70 75 80 HisGlu Asp Leu Glu Asn Leu Thr Thr Tyr Gly Phe Arg Gly Glu Ala 85 90 95 LeuGly Ser Ile Cys Cys Ile Ala Glu Val Leu Ile Thr Thr Arg Thr 100 105 110Ala Ala Asp Asn Phe Ser Thr Gln Tyr Val Leu Asp Gly Ser Gly His 115 120125 Ile Leu Ser Gln Lys 130 14 426 DNA Homo sapiens 14 cgaggcggatcgggtgttgc atccatggag cgagctgaga gctcgagtac agaacctgct 60 aaggccatcaaacctattga tcggaagtca gtccatcaga tttgctctgg gcaggtggta 120 ctgagtctaagcactgcggt aaaggagtta gtagaaaaca gtctggatgc tggtgccact 180 aatattgatctaaagcttaa ggactatgga gtggatctta ttgaagtttc agacaatgga 240 tgtggggtagaagaagaaaa cttcgaaggc ttaactctga aacatcacac atctaagatt 300 caagagtttgccgacctaac tcaggttgaa acttttggct ttcgggggga agctctgagc 360 tcactttgtgcactgagcga tgtcaccatt tctacctgcc acgcatcggc gaaggttgga 420 acttga 426 151360 PRT Homo sapiens 15 Met Ser Arg Gln Ser Thr Leu Tyr Ser Phe Phe ProLys Ser Pro Ala 1 5 10 15 Leu Ser Asp Ala Asn Lys Ala Ser Ala Arg AlaSer Arg Glu Gly Gly 20 25 30 Arg Ala Ala Ala Ala Pro Gly Ala Ser Pro SerPro Gly Gly Asp Ala 35 40 45 Ala Trp Ser Glu Ala Gly Pro Gly Pro Arg ProLeu Ala Arg Ser Ala 50 55 60 Ser Pro Pro Lys Ala Lys Asn Leu Asn Gly GlyLeu Arg Arg Ser Val 65 70 75 80 Ala Pro Ala Ala Pro Thr Ser Cys Asp PheSer Pro Gly Asp Leu Val 85 90 95 Trp Ala Lys Met Glu Gly Tyr Pro Trp TrpPro Cys Leu Val Tyr Asn 100 105 110 His Pro Phe Asp Gly Thr Phe Ile ArgGlu Lys Gly Lys Ser Val Arg 115 120 125 Val His Val Gln Phe Phe Asp AspSer Pro Thr Arg Gly Trp Val Ser 130 135 140 Lys Arg Leu Leu Lys Pro TyrThr Gly Ser Lys Ser Lys Glu Ala Gln 145 150 155 160 Lys Gly Gly His PheTyr Ser Ala Lys Pro Glu Ile Leu Arg Ala Met 165 170 175 Gln Arg Ala AspGlu Ala Leu Asn Lys Asp Lys Ile Lys Arg Leu Glu 180 185 190 Leu Ala ValCys Asp Glu Pro Ser Glu Pro Glu Glu Glu Glu Glu Met 195 200 205 Glu ValGly Thr Thr Tyr Val Thr Asp Lys Ser Glu Glu Asp Asn Glu 210 215 220 IleGlu Ser Glu Glu Glu Val Gln Pro Lys Thr Gln Gly Ser Arg Arg 225 230 235240 Ser Ser Arg Gln Ile Lys Lys Arg Arg Val Ile Ser Asp Ser Glu Ser 245250 255 Asp Ile Gly Gly Ser Asp Val Glu Phe Lys Pro Asp Thr Lys Glu Glu260 265 270 Gly Ser Ser Asp Glu Ile Ser Ser Gly Val Gly Asp Ser Glu SerGlu 275 280 285 Gly Leu Asn Ser Pro Val Lys Val Ala Arg Lys Arg Lys ArgMet Val 290 295 300 Thr Gly Asn Gly Ser Leu Lys Arg Lys Ser Ser Arg LysGlu Thr Pro 305 310 315 320 Ser Ala Thr Lys Gln Ala Thr Ser Ile Ser SerGlu Thr Lys Asn Thr 325 330 335 Leu Arg Ala Phe Ser Ala Pro Gln Asn SerGlu Ser Gln Ala His Val 340 345 350 Ser Gly Gly Gly Asp Asp Ser Ser ArgPro Thr Val Trp Tyr His Glu 355 360 365 Thr Leu Glu Trp Leu Lys Glu GluLys Arg Arg Asp Glu His Arg Arg 370 375 380 Arg Pro Asp His Pro Asp PheAsp Ala Ser Thr Leu Tyr Val Pro Glu 385 390 395 400 Asp Phe Leu Asn SerCys Thr Pro Gly Met Arg Lys Trp Trp Gln Ile 405 410 415 Lys Ser Gln AsnPhe Asp Leu Val Ile Cys Tyr Lys Val Gly Lys Phe 420 425 430 Tyr Glu LeuTyr His Met Asp Ala Leu Ile Gly Val Ser Glu Leu Gly 435 440 445 Leu ValPhe Met Lys Gly Asn Trp Ala His Ser Gly Phe Pro Glu Ile 450 455 460 AlaPhe Gly Arg Tyr Ser Asp Ser Leu Val Gln Lys Gly Tyr Lys Val 465 470 475480 Ala Arg Val Glu Gln Thr Glu Thr Pro Glu Met Met Glu Ala Arg Cys 485490 495 Arg Lys Met Ala His Ile Ser Lys Tyr Asp Arg Val Val Arg Arg Glu500 505 510 Ile Cys Arg Ile Ile Thr Lys Gly Thr Gln Thr Tyr Ser Val LeuGlu 515 520 525 Gly Asp Pro Ser Glu Asn Tyr Ser Lys Tyr Leu Leu Ser LeuLys Glu 530 535 540 Lys Glu Glu Asp Ser Ser Gly His Thr Arg Ala Tyr GlyVal Cys Phe 545 550 555 560 Val Asp Thr Ser Leu Gly Lys Phe Phe Ile GlyGln Phe Ser Asp Asp 565 570 575 Arg His Cys Ser Arg Phe Arg Thr Leu ValAla His Tyr Pro Pro Val 580 585 590 Gln Val Leu Phe Glu Lys Gly Asn LeuSer Lys Glu Thr Lys Thr Ile 595 600 605 Leu Lys Ser Ser Leu Ser Cys SerLeu Gln Glu Gly Leu Ile Pro Gly 610 615 620 Ser Gln Phe Trp Asp Ala SerLys Thr Leu Arg Thr Leu Leu Glu Glu 625 630 635 640 Glu Tyr Phe Arg GluLys Leu Ser Asp Gly Ile Gly Val Met Leu Pro 645 650 655 Gln Val Leu LysGly Met Thr Ser Glu Ser Asp Ser Ile Gly Leu Thr 660 665 670 Pro Gly GluLys Ser Glu Leu Ala Leu Ser Ala Leu Gly Gly Cys Val 675 680 685 Phe TyrLeu Lys Lys Cys Leu Ile Asp Gln Glu Leu Leu Ser Met Ala 690 695 700 AsnPhe Glu Glu Tyr Ile Pro Leu Asp Ser Asp Thr Val Ser Thr Thr 705 710 715720 Arg Ser Gly Ala Ile Phe Thr Lys Ala Tyr Gln Arg Met Val Leu Asp 725730 735 Ala Val Thr Leu Asn Asn Leu Glu Ile Phe Leu Asn Gly Thr Asn Gly740 745 750 Ser Thr Glu Gly Thr Leu Leu Glu Arg Val Asp Thr Cys His ThrPro 755 760 765 Phe Gly Lys Arg Leu Leu Lys Gln Trp Leu Cys Ala Pro LeuCys Asn 770 775 780 His Tyr Ala Ile Asn Asp Arg Leu Asp Ala Ile Glu AspLeu Met Val 785 790 795 800 Val Pro Asp Lys Ile Ser Glu Val Val Glu LeuLeu Lys Lys Leu Pro 805 810 815 Asp Leu Glu Arg Leu Leu Ser Lys Ile HisAsn Val Gly Ser Pro Leu 820 825 830 Lys Ser Gln Asn His Pro Asp Ser ArgAla Ile Met Tyr Glu Glu Thr 835 840 845 Thr Tyr Ser Lys Lys Lys Ile IleAsp Phe Leu Ser Ala Leu Glu Gly 850 855 860 Phe Lys Val Met Cys Lys IleIle Gly Ile Met Glu Glu Val Ala Asp 865 870 875 880 Gly Phe Lys Ser LysIle Leu Lys Gln Val Ile Ser Leu Gln Thr Lys 885 890 895 Asn Pro Glu GlyArg Phe Pro Asp Leu Thr Val Glu Leu Asn Arg Trp 900 905 910 Asp Thr AlaPhe Asp His Glu Lys Ala Arg Lys Thr Gly Leu Ile Thr 915 920 925 Pro LysAla Gly Phe Asp Ser Asp Tyr Asp Gln Ala Leu Ala Asp Ile 930 935 940 ArgGlu Asn Glu Gln Ser Leu Leu Glu Tyr Leu Glu Lys Gln Arg Asn 945 950 955960 Arg Ile Gly Cys Arg Thr Ile Val Tyr Trp Gly Ile Gly Arg Asn Arg 965970 975 Tyr Gln Leu Glu Ile Pro Glu Asn Phe Thr Thr Arg Asn Leu Pro Glu980 985 990 Glu Tyr Glu Leu Lys Ser Thr Lys Lys Gly Cys Lys Arg Tyr TrpThr 995 1000 1005 Lys Thr Ile Glu Lys Lys Leu Ala Asn Leu Ile Asn AlaGlu Glu 1010 1015 1020 Arg Arg Asp Val Ser Leu Lys Asp Cys Met Arg ArgLeu Phe Tyr 1025 1030 1035 Asn Phe Asp Lys Asn Tyr Lys Asp Trp Gln SerAla Val Glu Cys 1040 1045 1050 Ile Ala Val Leu Asp Val Leu Leu Cys LeuAla Asn Tyr Ser Arg 1055 1060 1065 Gly Gly Asp Gly Pro Met Cys Arg ProVal Ile Leu Leu Pro Glu 1070 1075 1080 Asp Thr Pro Pro Phe Leu Glu LeuLys Gly Ser Arg His Pro Cys 1085 1090 1095 Ile Thr Lys Thr Phe Phe GlyAsp Asp Phe Ile Pro Asn Asp Ile 1100 1105 1110 Leu Ile Gly Cys Glu GluGlu Glu Gln Glu Asn Gly Lys Ala Tyr 1115 1120 1125 Cys Val Leu Val ThrGly Pro Asn Met Gly Gly Lys Ser Thr Leu 1130 1135 1140 Met Arg Gln AlaGly Leu Leu Ala Val Met Ala Gln Met Gly Cys 1145 1150 1155 Tyr Val ProAla Glu Val Cys Arg Leu Thr Pro Ile Asp Arg Val 1160 1165 1170 Phe ThrArg Leu Gly Ala Ser Asp Arg Ile Met Ser Gly Glu Ser 1175 1180 1185 ThrPhe Phe Val Glu Leu Ser Glu Thr Ala Ser Ile Leu Met His 1190 1195 1200Ala Thr Ala His Ser Leu Val Leu Val Asp Glu Leu Gly Arg Gly 1205 12101215 Thr Ala Thr Phe Asp Gly Thr Ala Ile Ala Asn Ala Val Val Lys 12201225 1230 Glu Leu Ala Glu Thr Ile Lys Cys Arg Thr Leu Phe Ser Thr His1235 1240 1245 Tyr His Ser Leu Val Glu Asp Tyr Ser Gln Asn Val Ala ValArg 1250 1255 1260 Leu Gly His Met Ala Cys Met Val Glu Asn Glu Cys GluAsp Pro 1265 1270 1275 Ser Gln Glu Thr Ile Thr Phe Leu Tyr Lys Phe IleLys Gly Ala 1280 1285 1290 Cys Pro Lys Ser Tyr Gly Phe Asn Ala Ala ArgLeu Ala Asn Leu 1295 1300 1305 Pro Glu Glu Val Ile Gln Lys Gly His ArgLys Ala Arg Glu Phe 1310 1315 1320 Glu Lys Met Asn Gln Ser Leu Arg LeuPhe Arg Glu Val Cys Leu 1325 1330 1335 Ala Ser Glu Arg Ser Thr Val AspAla Glu Ala Val His Lys Leu 1340 1345 1350 Leu Thr Leu Ile Lys Glu Leu1355 1360 16 4264 DNA Homo sapiens 16 atttcccgcc agcaggagcc gcgcggtagatgcggtgctt ttaggagctc cgtccgacag 60 aacggttggg ccttgccggc tgtcggtatgtcgcgacaga gcaccctgta cagcttcttc 120 cccaagtctc cggcgctgag tgatgccaacaaggcctcgg ccagggcctc acgcgaaggc 180 ggccgtgccg ccgctgcccc cggggcctctccttccccag gcggggatgc ggcctggagc 240 gaggctgggc ctgggcccag gcccttggcgcgatccgcgt caccgcccaa ggcgaagaac 300 ctcaacggag ggctgcggag atcggtagcgcctgctgccc ccaccagttg tgacttctca 360 ccaggagatt tggtttgggc caagatggagggttacccct ggtggccttg tctggtttac 420 aaccacccct ttgatggaac attcatccgcgagaaaggga aatcagtccg tgttcatgta 480 cagttttttg atgacagccc aacaaggggctgggttagca aaaggctttt aaagccatat 540 acaggttcaa aatcaaagga agcccagaagggaggtcatt tttacagtgc aaagcctgaa 600 atactgagag caatgcaacg tgcagatgaagccttaaata aagacaagat taagaggctt 660 gaattggcag tttgtgatga gccctcagagccagaagagg aagaagagat ggaggtaggc 720 acaacttacg taacagataa gagtgaagaagataatgaaa ttgagagtga agaggaagta 780 cagcctaaga cacaaggatc taggcgaagtagccgccaaa taaaaaaacg aagggtcata 840 tcagattctg agagtgacat tggtggctctgatgtggaat ttaagccaga cactaaggag 900 gaaggaagca gtgatgaaat aagcagtggagtgggggata gtgagagtga aggcctgaac 960 agccctgtca aagttgctcg aaagcggaagagaatggtga ctggaaatgg ctctcttaaa 1020 aggaaaagct ctaggaagga aacgccctcagccaccaaac aagcaactag catttcatca 1080 gaaaccaaga atactttgag agctttctctgcccctcaaa attctgaatc ccaagcccac 1140 gttagtggag gtggtgatga cagtagtcgccctactgttt ggtatcatga aactttagaa 1200 tggcttaagg aggaaaagag aagagatgagcacaggagga ggcctgatca ccccgatttt 1260 gatgcatcta cactctatgt gcctgaggatttcctcaatt cttgtactcc tgggatgagg 1320 aagtggtggc agattaagtc tcagaactttgatcttgtca tctgttacaa ggtggggaaa 1380 ttttatgagc tgtaccacat ggatgctcttattggagtca gtgaactggg gctggtattc 1440 atgaaaggca actgggccca ttctggctttcctgaaattg catttggccg ttattcagat 1500 tccctggtgc agaagggcta taaagtagcacgagtggaac agactgagac tccagaaatg 1560 atggaggcac gatgtagaaa gatggcacatatatccaagt atgatagagt ggtgaggagg 1620 gagatctgta ggatcattac caagggtacacagacttaca gtgtgctgga aggtgatccc 1680 tctgagaact acagtaagta tcttcttagcctcaaagaaa aagaggaaga ttcttctggc 1740 catactcgtg catatggtgt gtgctttgttgatacttcac tgggaaagtt tttcataggt 1800 cagttttcag atgatcgcca ttgttcgagatttaggactc tagtggcaca ctatccccca 1860 gtacaagttt tatttgaaaa aggaaatctctcaaaggaaa ctaaaacaat tctaaagagt 1920 tcattgtcct gttctcttca ggaaggtctgatacccggct cccagttttg ggatgcatcc 1980 aaaactttga gaactctcct tgaggaagaatattttaggg aaaagctaag tgatggcatt 2040 ggggtgatgt taccccaggt gcttaaaggtatgacttcag agtctgattc cattgggttg 2100 acaccaggag agaaaagtga attggccctctctgctctag gtggttgtgt cttctacctc 2160 aaaaaatgcc ttattgatca ggagcttttatcaatggcta attttgaaga atatattccc 2220 ttggattctg acacagtcag cactacaagatctggtgcta tcttcaccaa agcctatcaa 2280 cgaatggtgc tagatgcagt gacattaaacaacttggaga tttttctgaa tggaacaaat 2340 ggttctactg aaggaaccct actagagagggttgatactt gccatactcc ttttggtaag 2400 cggctcctaa agcaatggct ttgtgccccactctgtaacc attatgctat taatgatcgt 2460 ctagatgcca tagaagacct catggttgtgcctgacaaaa tctccgaagt tgtagagctt 2520 ctaaagaagc ttccagatct tgagaggctactcagtaaaa ttcataatgt tgggtctccc 2580 ctgaagagtc agaaccaccc agacagcagggctataatgt atgaagaaac tacatacagc 2640 aagaagaaga ttattgattt tctttctgctctggaaggat tcaaagtaat gtgtaaaatt 2700 atagggatca tggaagaagt tgctgatggttttaagtcta aaatccttaa gcaggtcatc 2760 tctctgcaga caaaaaatcc tgaaggtcgttttcctgatt tgactgtaga attgaaccga 2820 tgggatacag cctttgacca tgaaaaggctcgaaagactg gacttattac tcccaaagca 2880 ggctttgact ctgattatga ccaagctcttgctgacataa gagaaaatga acagagcctc 2940 ctggaatacc tagagaaaca gcgcaacagaattggctgta ggaccatagt ctattggggg 3000 attggtagga accgttacca gctggaaattcctgagaatt tcaccactcg caatttgcca 3060 gaagaatacg agttgaaatc taccaagaagggctgtaaac gatactggac caaaactatt 3120 gaaaagaagt tggctaatct cataaatgctgaagaacgga gggatgtatc attgaaggac 3180 tgcatgcggc gactgttcta taactttgataaaaattaca aggactggca gtctgctgta 3240 gagtgtatcg cagtgttgga tgttttactgtgcctggcta actatagtcg agggggtgat 3300 ggtcctatgt gtcgcccagt aattctgttgccggaagata cccccccctt cttagagctt 3360 aaaggatcac gccatccttg cattacgaagactttttttg gagatgattt tattcctaat 3420 gacattctaa taggctgtga ggaagaggagcaggaaaatg gcaaagccta ttgtgtgctt 3480 gttactggac caaatatggg gggcaagtctacgcttatga gacaggctgg cttattagct 3540 gtaatggccc agatgggttg ttacgtccctgctgaagtgt gcaggctcac accaattgat 3600 agagtgttta ctagacttgg tgcctcagacagaataatgt caggtgaaag tacatttttt 3660 gttgaattaa gtgaaactgc cagcatactcatgcatgcaa cagcacattc tctggtgctt 3720 gtggatgaat taggaagagg tactgcaacatttgatggga cggcaatagc aaatgcagtt 3780 gttaaagaac ttgctgagac tataaaatgtcgtacattat tttcaactca ctaccattca 3840 ttagtagaag attattctca aaatgttgctgtgcgcctag gacatatggc atgcatggta 3900 gaaaatgaat gtgaagaccc cagccaggagactattacgt tcctctataa attcattaag 3960 ggagcttgtc ctaaaagcta tggctttaatgcagcaaggc ttgctaatct cccagaggaa 4020 gttattcaaa agggacatag aaaagcaagagaatttgaga agatgaatca gtcactacga 4080 ttatttcggg aagtttgcct ggctagtgaaaggtcaactg tagatgctga agctgtccat 4140 aaattgctga ctttgattaa ggaattatagactgactaca ttggaagctt tgagttgact 4200 tctgaccaaa ggtggtaaat tcagacaacattatgatcta ataaacttta ttttttaaaa 4260 atga 4264 17 1408 DNA Homo sapiens17 ggcgctccta cctgcaagtg gctagtgcca agtgctgggc cgccgctcct gccgtgcatg 60ttggggagcc agtacatgca ggtgggctcc acacggagag gggcgcagac ccggtgacag 120ggctttacct ggtacatcgg catggcgcaa ccaaagcaag agagggtggc gcgtgccaga 180caccaacggt cggaaaccgc cagacaccaa cggtcggaaa ccgccaagac accaacgctc 240ggaaaccgcc agacaccaac gctcggaaac cgccagacac caaggctcgg aatccacgcc 300aggccacgac ggagggcgac tacctccctt ctgaccctgc tgctggcgtt cggaaaaaac 360gcagtccggt gtgctctgat tggtccaggc tctttgacgt cacggactcg acctttgaca 420gagccactag gcgaaaagga gagacgggaa gtattttttc cgccccgccc ggaaagggtg 480gagcacaacg tcgaaagcag ccgttgggag cccaggaggc ggggcgcctg tgggagccgt 540ggagggaact ttcccagtcc ccgaggcgga tccggtgttg catccttgga gcgagctgag 600aactcgagta cagaacctgc taaggccatc aaacctattg atcggaagtc agtccatcag 660atttgctctg ggccggtggt accgagtcta aggccgaatg cggtgaagga gttagtagaa 720aacagtctgg atgctggtgc cactaatgtt gatctaaagc ttaaggacta tggagtggat 780ctcattgaag tttcaggcaa tggatgtggg gtagaagaag aaaacttcga aggctttact 840ctgaaacatc acacatgtaa gattcaagag tttgccgacc taactcaggt ggaaactttt 900ggctttcggg gggaagctct gagctcactt tgtgcactga gtgatgtcac catttctacc 960tgccgtgtat cagcgaaggt tgggactcga ctggtgtttg atcactatgg gaaaatcatc 1020cagaaaaccc cctacccccg ccccagaggg atgacagtca gcgtgaagca gttattttct 1080acgctacctg tgcaccataa agaatttcaa aggaatatta agaagaaacg tgcctgcttc 1140cccttcgcct tctgccgtga ttgtcagttt cctgaggcct ccccagccat gcttcctgta 1200cagcctgtag aactgactcc tagaagtacc ccaccccacc cctgctcctt ggaggacaac 1260gtgatcactg tattcagctc tgtcaagaat ggtccaggtt cttctagatg atctgcacaa 1320atggttcctc tcctccttcc tgatgtctgc cattagcatt ggaataaagt tcctgctgaa 1380aatccaaaaa aaaaaaaaaa aaaaaaaa 1408 18 389 PRT Homo sapiens 18 Met AlaGln Pro Lys Gln Glu Arg Val Ala Arg Ala Arg His Gln Arg 1 5 10 15 SerGlu Thr Ala Arg His Gln Arg Ser Glu Thr Ala Lys Thr Pro Thr 20 25 30 LeuGly Asn Arg Gln Thr Pro Thr Leu Gly Asn Arg Gln Thr Pro Arg 35 40 45 LeuGly Ile His Ala Arg Pro Arg Arg Arg Ala Thr Thr Ser Leu Leu 50 55 60 ThrLeu Leu Leu Ala Phe Gly Lys Asn Ala Val Arg Cys Ala Leu Ile 65 70 75 80Gly Pro Gly Ser Leu Thr Ser Arg Thr Arg Pro Leu Thr Glu Pro Leu 85 90 95Gly Glu Lys Glu Arg Arg Glu Val Phe Phe Pro Pro Arg Pro Glu Arg 100 105110 Val Glu His Asn Val Glu Ser Ser Arg Trp Glu Pro Arg Arg Arg Gly 115120 125 Ala Cys Gly Ser Arg Gly Gly Asn Phe Pro Ser Pro Arg Gly Gly Ser130 135 140 Gly Val Ala Ser Leu Glu Arg Ala Glu Asn Ser Ser Thr Glu ProAla 145 150 155 160 Lys Ala Ile Lys Pro Ile Asp Arg Lys Ser Val His GlnIle Cys Ser 165 170 175 Gly Pro Val Val Pro Ser Leu Arg Pro Asn Ala ValLys Glu Leu Val 180 185 190 Glu Asn Ser Leu Asp Ala Gly Ala Thr Asn ValAsp Leu Lys Leu Lys 195 200 205 Asp Tyr Gly Val Asp Leu Ile Glu Val SerGly Asn Gly Cys Gly Val 210 215 220 Glu Glu Glu Asn Phe Glu Gly Phe ThrLeu Lys His His Thr Cys Lys 225 230 235 240 Ile Gln Glu Phe Ala Asp LeuThr Gln Val Glu Thr Phe Gly Phe Arg 245 250 255 Gly Glu Ala Leu Ser SerLeu Cys Ala Leu Ser Asp Val Thr Ile Ser 260 265 270 Thr Cys Arg Val SerAla Lys Val Gly Thr Arg Leu Val Phe Asp His 275 280 285 Tyr Gly Lys IleIle Gln Lys Thr Pro Tyr Pro Arg Pro Arg Gly Met 290 295 300 Thr Val SerVal Lys Gln Leu Phe Ser Thr Leu Pro Val His His Lys 305 310 315 320 GluPhe Gln Arg Asn Ile Lys Lys Lys Arg Ala Cys Phe Pro Phe Ala 325 330 335Phe Cys Arg Asp Cys Gln Phe Pro Glu Ala Ser Pro Ala Met Leu Pro 340 345350 Val Gln Pro Val Glu Leu Thr Pro Arg Ser Thr Pro Pro His Pro Cys 355360 365 Ser Leu Glu Asp Asn Val Ile Thr Val Phe Ser Ser Val Lys Asn Gly370 375 380 Pro Gly Ser Ser Arg 385 19 795 DNA Homo sapiens 19atgtgtcctt ggcggcctag actaggccgt cgctgtatgg tgagccccag ggaggcggat 60ctgggccccc agaaggacac ccgcctggat ttgccccgta gcccggcccg ggcccctcgg 120gagcagaaca gccttggtga ggtggacagg aggggacctc gcgagcagac gcgcgcgcca 180gcgacagcag ccccgccccg gcctctcggg agccgggggg cagaggctgc ggagccccag 240gagggtctat cagccacagt ctctgcatgt ttccaagagc aacaggaaat gaacacattg 300caggggccag tgtcattcaa agatgtggct gtggatttca cccaggagga gtggcggcaa 360ctggaccctg atgagaagat agcatacggg gatgtgatgt tggagaacta cagccatcta 420gtttctgtgg ggtatgatta tcaccaagcc aaacatcatc atggagtgga ggtgaaggaa 480gtggagcagg gagaggagcc gtggataatg gaaggtgaat ttccatgtca acatagtcca 540gaacctgcta aggccatcaa acctattgat cggaagtcag tccatcagat ttgctctggg 600ccagtggtac tgagtctaag cactgcagtg aaggagttag tagaaaacag tctggatgct 660ggtgccacta atattgatct aaagcttaag gactatggag tggatctcat tgaagtttca 720gacaatggat gtggggtaga agaagaaaac tttgaaggct taatctcttt cagctctgaa 780acatcacaca tgtaa 795 20 264 PRT Homo sapiens 20 Met Cys Pro Trp Arg ProArg Leu Gly Arg Arg Cys Met Val Ser Pro 1 5 10 15 Arg Glu Ala Asp LeuGly Pro Gln Lys Asp Thr Arg Leu Asp Leu Pro 20 25 30 Arg Ser Pro Ala ArgAla Pro Arg Glu Gln Asn Ser Leu Gly Glu Val 35 40 45 Asp Arg Arg Gly ProArg Glu Gln Thr Arg Ala Pro Ala Thr Ala Ala 50 55 60 Pro Pro Arg Pro LeuGly Ser Arg Gly Ala Glu Ala Ala Glu Pro Gln 65 70 75 80 Glu Gly Leu SerAla Thr Val Ser Ala Cys Phe Gln Glu Gln Gln Glu 85 90 95 Met Asn Thr LeuGln Gly Pro Val Ser Phe Lys Asp Val Ala Val Asp 100 105 110 Phe Thr GlnGlu Glu Trp Arg Gln Leu Asp Pro Asp Glu Lys Ile Ala 115 120 125 Tyr GlyAsp Val Met Leu Glu Asn Tyr Ser His Leu Val Ser Val Gly 130 135 140 TyrAsp Tyr His Gln Ala Lys His His His Gly Val Glu Val Lys Glu 145 150 155160 Val Glu Gln Gly Glu Glu Pro Trp Ile Met Glu Gly Glu Phe Pro Cys 165170 175 Gln His Ser Pro Glu Pro Ala Lys Ala Ile Lys Pro Ile Asp Arg Lys180 185 190 Ser Val His Gln Ile Cys Ser Gly Pro Val Val Leu Ser Leu SerThr 195 200 205 Ala Val Lys Glu Leu Val Glu Asn Ser Leu Asp Ala Gly AlaThr Asn 210 215 220 Ile Asp Leu Lys Leu Lys Asp Tyr Gly Val Asp Leu IleGlu Val Ser 225 230 235 240 Asp Asn Gly Cys Gly Val Glu Glu Glu Asn PheGlu Gly Leu Ile Ser 245 250 255 Phe Ser Ser Glu Thr Ser His Met 260 211445 DNA Homo sapiens 21 tttttttttt tgatgttctc cagtgcctca gtggcagcagaactggccct gtatcaggcc 60 gctaccgcca ctccatgacc aacctccctg catacccccccccccagcac ccctcccaca 120 ggaccgcttc tgtgtttggg acccaccagg cctttgcaccatacaacaaa ccctcactct 180 ccggggcccg gtctgcgccc aggctgaaca ccacgaacgcctgggacgca gctcctcctt 240 ccctggggag ccagcccctc taccgctcca gcctctcccacctgggaccg cagcacctgc 300 ccccaggatc ctccacctcc ggtgcagtca gtgcctccctccccagcggt ccctcaagca 360 gcccaggcga gcgtccctgc cactgtgccc atgcagatgccaagccagca gagtcagcag 420 gcgctcgctg gagcgacccg aagccagagc agagcagagcaggtcataaa actacacgga 480 agagctgaaa gtgcccccag atgaggactg catcatctgcatggagaagc tgtccgcagc 540 gtctggatac agcgatgtga ctgacagcaa ggcaatggggcccctggctg tgggctgcct 600 caccaagtgc agccacgcct tccacctgct gtgcctcctggccatgtact gcaacggcaa 660 taagggccct gagcacccca atcccggaaa gccgttcactgccagagggt ttcccgccag 720 tgctaccttc cagacaacgc cagggccgca agcctccaggggcttccaga acccggagac 780 actggctgac attccggcct ccccacagct gctgaccgatggccactaca tgacgctgcc 840 cgtgtctccg gaccagctgc cctgtgacga ccccatggcgggcagcggag gcgcccccgt 900 gctgcgggtg ggccatgacc acggctgcca ccagcagccacgtatctgca acgcgcccct 960 ccctggccct ggaccctatc gtacagaacc tgctaaggccatcaaaccta ttgatcggaa 1020 gtcagtccat cagatttgct ctgggccagt ggtactgagtctaagcactg cagtgaagga 1080 gttagtagaa aacagtctgg atgctggtgc cactaatattgatctaaagc ttaaggacta 1140 tggaatggat ctcattgaag tttcaggcaa tggatgtggggtagaagaag aaaacttcga 1200 aggcttaatg atgtcaccat ttctacctgc cacgtctcggcgaaggttgg gactcgactg 1260 gtgtttgatc acgatgggaa aatcatccag aagaccccctacccccaccc cagagggacc 1320 acagtcagcg tgaagcagtt attttctacg ctacctgtgcgccataagga atttcaaagg 1380 aatattaaga agaaacatgc tgcttcccct tcgccttctgccgtgattgt cagttttaac 1440 cggaa 1445 22 270 PRT Homo sapiens 22 Met GluLys Leu Ser Ala Ala Ser Gly Tyr Ser Asp Val Thr Asp Ser 1 5 10 15 LysAla Met Gly Pro Leu Ala Val Gly Cys Leu Thr Lys Cys Ser His 20 25 30 AlaPhe His Leu Leu Cys Leu Leu Ala Met Tyr Cys Asn Gly Asn Lys 35 40 45 GlyPro Glu His Pro Asn Pro Gly Lys Pro Phe Thr Ala Arg Gly Phe 50 55 60 ProAla Ser Ala Thr Phe Gln Thr Thr Pro Gly Pro Gln Ala Ser Arg 65 70 75 80Gly Phe Gln Asn Pro Glu Thr Leu Ala Asp Ile Pro Ala Ser Pro Gln 85 90 95Leu Leu Thr Asp Gly His Tyr Met Thr Leu Pro Val Ser Pro Asp Gln 100 105110 Leu Pro Cys Asp Asp Pro Met Ala Gly Ser Gly Gly Ala Pro Val Leu 115120 125 Arg Val Gly His Asp His Gly Cys His Gln Gln Pro Arg Ile Cys Asn130 135 140 Ala Pro Leu Pro Gly Pro Gly Pro Tyr Arg Thr Glu Pro Ala LysAla 145 150 155 160 Ile Lys Pro Ile Asp Arg Lys Ser Val His Gln Ile CysSer Gly Pro 165 170 175 Val Val Leu Ser Leu Ser Thr Ala Val Lys Glu LeuVal Glu Asn Ser 180 185 190 Leu Asp Ala Gly Ala Thr Asn Ile Asp Leu LysLeu Lys Asp Tyr Gly 195 200 205 Met Asp Leu Ile Glu Val Ser Gly Asn GlyCys Gly Val Glu Glu Glu 210 215 220 Asn Phe Glu Gly Leu Met Met Ser ProPhe Leu Pro Ala Thr Ser Arg 225 230 235 240 Arg Arg Leu Gly Leu Asp TrpCys Leu Ile Thr Met Gly Lys Ser Ser 245 250 255 Arg Arg Pro Pro Thr ProThr Pro Glu Gly Pro Gln Ser Ala 260 265 270 23 16 PRT Homo sapiens 23Ala Val Lys Glu Leu Val Glu Asn Ser Leu Asp Ala Gly Ala Thr Asn 1 5 1015 24 48 PRT Homo sapiens 24 Leu Arg Pro Asn Ala Val Lys Glu Leu Val GluAsn Ser Leu Asp Ala 1 5 10 15 Gly Ala Thr Asn Val Asp Leu Lys Leu LysAsp Tyr Gly Val Asp Leu 20 25 30 Ile Glu Val Ser Gly Asn Gly Cys Gly ValGlu Glu Glu Asn Phe Glu 35 40 45 25 47 PRT Homo sapiens 25 Leu Ser ThrAla Val Lys Glu Leu Val Glu Asn Ser Leu Asp Ala Gly 1 5 10 15 Ala ThrAsn Ile Asp Leu Lys Leu Lys Asp Tyr Gly Val Asp Leu Ile 20 25 30 Glu ValSer Asp Asn Gly Cys Gly Val Glu Glu Glu Asn Phe Glu 35 40 45 26 50 PRTHomo sapiens 26 Leu Arg Gln Val Leu Ser Asn Leu Leu Asp Asn Ala Ile LysTyr Thr 1 5 10 15 Pro Glu Gly Gly Glu Ile Thr Val Ser Leu Glu Arg AspGly Asp His 20 25 30 Leu Glu Ile Thr Val Glu Asp Asn Gly Pro Gly Ile ProGlu Glu Asp 35 40 45 Leu Glu 50 27 22 DNA artificial Oligonucleotideprimer 27 ggacgagaag tataacttcg ag 22 28 21 DNA ArtificialOligonucleotide primer 28 catctcgctt gtgttaagag c 21 29 19 DNAArtificial Oligonucleotide primer 29 ggcgcaacca aagcaagag 19 30 19 DNAArtificial Oligonucleotide primer 30 actgcgtttt ttccgaacg 19 31 19 DNAArtificial Oligonucleotide primer 31 atgttggaga actacagcc 19 32 19 DNAArtificial Oligonucleotide primer 32 cactccatag tccttaagc 19 33 19 DNAArtificial Oligonucleotide primer 33 gggaatgggt cagaaggac 19 34 20 DNAArtificial Oligonucleotide primer 34 tttcacggtt ggccttaggg 20 35 21 DNAArtificial Oligonucleotide primer 35 tgactacttt tgacttcagc c 21 36 22DNA Artificial Oligonucleotide primer 36 aaccattcaa catttttaac cc 22 3721 DNA Artificial Oligonucleotide primer 37 attaacttcc tacaccacaa c 2138 19 DNA Artificial Oligonucleotide primer 38 gtagagcaag accaccttg 1939 20 DNA Artificial Oligonucleotide primer 39 acattgctgg aagttctggc 2040 20 DNA Artificial Oligonucleotide primer 40 cctttctgac ttggatacca 2041 20 PRT Homo sapiens 41 Met Ala Gln Pro Lys Gln Glu Arg Val Ala ArgAla Arg His Gln Arg 1 5 10 15 Ser Glu Thr Ala 20 42 20 PRT Homo sapiens42 Leu Glu Asp Asn Val Ile Thr Val Phe Ser Ser Val Lys Asn Gly Pro 1 510 15 Gly Ser Ser Arg 20 43 20 PRT Homo sapiens 43 Arg Pro Arg Leu GlyArg Arg Cys Met Val Ser Pro Arg Ala Arg Ala 1 5 10 15 Pro Arg Glu Gln 2044 20 PRT Homo sapiens 44 Gly Val Glu Glu Glu Asn Phe Glu Gly Leu IleSer Phe Ser Ser Glu 1 5 10 15 Thr Ser His Met 20 45 30 DNA ArtificialOligonucleotide primer 45 acgcatatgg agcgagctga gagctcgagt 30 46 75 DNAArtificial Oligonucleotide primer 46 gaattcttat cacgtagaat cgagaccgaggagagggtta gggataggct taccagttcc 60 aaccttcgcc gatgc 75 47 27 DNAArtificial Oligonucleotide primer 47 acgcatatgt gtccttggcg gcctaga 27 4875 DNA Artificial Oligonucleotide primer 48 gaattcttat tacgtagaatcgagaccgag gagagggtta gggataggct tacccatgtg 60 tgatgtttca gagct 75

What is claimed is:
 1. A method of making a cell hypermutable comprisingintroducing into said cell a PMS2 homolog comprising a nucleotidesequence encoding a polypeptide comprising the amino acid sequence ofSEQ ID NO:23, thereby making a hypermutable cell, wherein said PMS2homolog is other than PMSR2 and PMSR3.
 2. The method of claim 1 whereinsaid polypeptide comprises the amino acid sequence of SEQ ID NO:24. 4.The method of claim 1 wherein said polypeptide comprises the amino acidsequence of SEQ ID NO:22.
 5. The method of claim 1 wherein said PMS2homolog encodes a protein having an ATPase domain.
 6. The method ofclaim 1 wherein said cell is a eukaryotic cell.
 7. The method of claim 1wherein said cell is a prokaryotic cell.
 8. The method of claim 6wherein said cell is a mammalian cell.
 9. The method of claim 8 whereinsaid cell is a human cell.
 10. The method of claim 1 further comprisingcontacting said cell with a mutagen.
 11. The method of claim 1 or 10further comprising screening said cell for a mutation in a gene ofinterest.
 12. The method of claim 11 wherein said screening is performedon the nucleic acid of said hypermutable cell.
 13. The method of claim11 wherein said screening is performed on the protein of saidhypermutable cell.
 14. The method of claim 11 wherein said screening isperformed by examining the phenotype of said hypermutable cell.
 15. Themethod of claim 11 further comprising restoring genetic stability ofsaid hypermutable cell.
 16. A method of making a mutation in a gene ofinterest comprising introducing into a cell containing a gene ofinterest a PMS2 homolog comprising a nucleotide sequence encoding apolypeptide comprising the amino acid sequence of SEQ ID NO:23, therebymaking said cell hypermutable, and selecting a mutant cell comprising amutation in said gene of interest.
 17. The method of claim 16 whereinsaid polypeptide comprises the amino acid sequence of SEQ ID NO:24. 18.The method of claim 16 wherein said polypeptide comprises the amino acidsequence of SEQ ID NO:22.
 19. The method of claim 16 wherein said PMS2homolog encodes a protein having an ATPase domain.
 20. The method ofclaim 16 wherein said cell is a eukaryotic cell.
 21. The method of claim16 wherein said cell is a prokaryotic cell.
 22. The method of claim 20wherein said cell is a mammalian cell.
 23. The method of claim 22wherein said cell is a human cell.
 24. The method of claim 16 furthercomprising contacting said cell with a mutagen.
 25. The method of claim16 or 24 further comprising restoring genetic stability of said mutantcell.
 26. A method of generating a library of mutant genes in a celltype comprising introducing into said cell type a PMS2 homologcomprising a nucleotide sequence encoding a polypeptide comprising theamino acid sequence of SEQ ID NO:23, thereby making hypermutable cells,wherein said PMS2 homolog is other than PMSR2 and PMSR3, incubating saidhypermutable cells type to allow mutations to accumulate, extractingnucleic acid from said hypermutable cells and creating a nucleic acidlibrary.
 27. The method of claim 26 wherein said polypeptide comprisesthe amino acid sequence of SEQ ID NO:24.
 28. The method of claim 27wherein said library is a cDNA library.
 29. The method of claim 27wherein said library is a genomic library.
 30. A method of assayingcells to detect neoplasia comprising contacting said sample with anucleotide sequence encoding the amino acid sequence of SEQ ID NO:23 todetect expression of a polynucleotide encoding a PMS2 homolog comprisingthe amino acid sequence of SEQ ID NO:23, wherein expression of said PMS2homolog is associated with neoplasia.
 31. The method of claim 30,wherein the detecting comprises a Northern blot analysis.
 32. The methodof claim 30, wherein the detecting comprises PCR.
 33. The method ofclaim 30, wherein detecting comprises RT-PCR analysis.
 34. A method ofassaying cells to detect neoplasia comprising contacting said samplewith an antibody directed against a PMS2 homolog or peptide fragmentsthereof; and detecting the presence of an antibody-complex formed withthe PMS2 homolog or peptide fragment thereof, thereby detecting thepresence of said PMS2 homolog in said sample, wherein the presence ofsaid PMS2 homolog is associated with neoplasia.
 35. The method of claim34, wherein the detecting comprises an immunoassay selected from thegroup consisting of a radioimmunoassay, a Western blot assay, animmunofluorescent assay, an enzyme-linked immunosorbent assay, and achemiluminescent assay.
 36. A method of treating a patient with cancercomprising identifying a patient with a PMS2 homolog-associatedneoplasm, administering to said patient an inhibitor of expression ofsaid PMS2 homolog wherein said inhibitor suppresses expression of saidPMS2 homolog in said PMS2 homolog associated neoplasm.
 37. The method ofclaim 36 wherein said PMS2 homolog associated neoplasm is a lymphoma.38. The method of claim 36 wherein said inhibitor of said PMS2 homologis an antisense nucleic acid directed against a polynucleotide encodingsaid PMS2 homolog.
 39. The method of claim 36 wherein said inhibitor ofsaid PMS2 homolog is a ribozyme.
 40. The method of claim 36 wherein saidinhibitor is a ATPase analog that specifically binds to said PMS2homolog.